No / Background Hyperhomocysteinaemia is an independent risk factor in the development of cardiovascular disease. Although homocysteine has been shown to affect endothelial cell function, the mechanisms by which it induces disease states are still poorly understood. Here, we report the ability of homocysteine to influence inflammatory cytokine/chemokine production by human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages. Methods Human saphenous vein endothelial cells, peripheral blood monocytes and monocyte-derived macrophages were treated with homocysteine (0.1-5 mmol/L) for 4 and/or 24 h. Tumour necrosis factor (TNF)-¿, interleukin (IL)-1ß, IL-6 and IL-8 production was measured in the cell culture media using commercially available enzyme-linked immunosorbent assays. Results Interleukin-6 production by human saphenous vein endothelial cells was significantly stimulated following a 24-h treatment with homocysteine, whilst IL-8 concentrations were inhibited after both 4- and 24-h treatments. Homocysteine was also found to stimulate IL-1ß production by human peripheral blood monocytes and TNF-¿ production by monocyte-derived macrophages. Conclusions Overall, results from this study suggest that homocysteine alters the profile of cytokine/chemokine production by endothelial cells and macrophages. This altered profile may be important in the inflammatory events that initiate or enhance the development of atherosclerotic lesions.

Yes / Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function. / Biotechnology and Biological Sciences Research Council, the Medical Research Council and the Wellcome Trust

BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

Cigarette smoke contributes to many diseases. The actions of second and third hand smoke, which have implications for non-smokers and the very young, are just beginning to be appreciated. The overarching hypothesis of this project is that cigarette smoke has different injurious actions on alveolar cells based on chronological age. The purpose here was to learn more about the susceptibility of alveolar cells to cigarette smoke extract (CSE)- induced injury by performing studies on pulmonary alveolar and endothelial cells derived from neonatal, young, and old rats. The aims involved: 1. Developing cell culture models to study age-related effects of cigarette smoke on alveolar type I cells and microvascular endothelial cells from the lung, and 2. Using these models to examine the effects of CSE on markers of oxidative stress, inflammation and aging in alveolar cells harvested from neonatal, young and old rats. Descriptive and experimental studies involved using a variety of cell culture, biochemical and molecular techniques, including gene expression arrays. The most significant findings were that: 1. primary proliferating alveolar type I cells were used to develop novel cell culture model systems, including single culture, co-culture and three-dimensional cultures that were used to study the effects of CSE; 2. Hydrogen peroxide production by endothelial cells was markedly reduced by co-culturing with AT I cells; 3. Gene expression profiling of oxidative stress-specific pathways suggest that genes responsible for both stopping production of H2O2 or mopping-up H2O2 are involved; and 4. Cigarette smoke shortens telomeres of cells from neonates, but unexpectedly preserves telomere length of cells from young and old rats. Data from telomeric pathway-specific gene expression arrays suggest that there are age-related differences in response to gene expression to CSE. The significant conclusions are: 1. Contrary to prior observations, alveolar type I cells demonstrate prolonged proliferative capacity; 2. Alveolar type I cells likely play an important role in ameliorating CSE-induced oxidative stress; and 3. Neonatal alveolar cells may be more susceptible to the deleterious effects of CSE including telomere shortening. These novel model systems and observations provide new ways to study cigarette smoke-associated lung dysfunction.

Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes.

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

Exercise training promotes a beneficial endothelial cell (EC) phenotype and results in conduit vessel adaptation. The specific underlying mechanisms have been proposed (shear stress, circumferential stress, hypoxia, metabolic) but are yet to be fully elucidated. This thesis investigated the predominant stimuli responsible for conduit vessel adaptation with training. Further, it developed a method of in situ EC extraction to allow for determination of the cellular and molecular mechanisms underpinning these adaptations. The methodology utilised two-dimensional (2D) Doppler ultrasound, strain gauge plethysmography, immunocytochemistry and RT-qPCR to provide insight in to vascular characteristics, predominantly of the brachial artery and peripheral EC. Long-term repeated isometric forearm muscle contractions as performed by well-trained rock climbers promoted greater resting, peak (in response to 5 min ischaemia) and maximal (in response to ischaemic exercise) brachial artery diameters compared with controls. This structural response is dependent upon confounders associated with exercise additional to shear stress as evidenced by the lack of brachial artery remodelling in response to 8 weeks of ischaemic preconditioning (IPC). A transient increase in flow-mediated dilation (FMD)% was observed following 6 weeks exposure to IPC, which became significant when controlled for baseline artery diameter, despite an absence of augmentation following long-term (≥ 8 weeks) exposure to a shear stimulus. This is in line with the suggested timeline of conduit vessel adaptation to exercise training of a transient increase in function at 2-4 weeks. Underpinning molecular mechanisms responsible were not determined but may be further investigated given that the endovascular biopsy technique was developed and improved in this thesis. The endovascular biopsy successfully yields approximately 2100 ± 1700 EC per sample, providing sufficient material for determination of expression of both mRNA (RT-qPCR) and protein (immunocytochemistry). Specifically, type 2 diabetics (T2DM) with symptomatic cardiac abnormalities exhibited augmented eNOS mRNA and protein in brachial artery EC as compared with non-diabetic controls with symptomatic cardiac abnormalities. In conclusion, this thesis demonstrates that although shear stress promotes a transient trend for enhancement in function of the peripheral conduit arteries, additional factors are required for long-term structural adaptations. Further, the endovascular biopsy technique offers a novel method of extracting and analysing EC for genes and proteins of interest to vascular health. The use of this technique to decipher the underlying cellular and molecular mechanisms involved in vascular adaptations with exercise requires further investigation.

A CTX estimula, in vivo e in vitro, a capacidade secretória de macrófagos na presença de tumor, acompanhado por significativa diminuição da massa tumoral ou proliferação de células tumorais, respectivamente. Considerando a ação imunomodulatória da CTX, a importância central do macrófago na gênese e progressão tumoral e a participação desta célula no microambiente estromal, a atividade secretória de macrófagos tratados com CTX sobre a angiogênese in vitro foi investigada. Células endoteliais tímicas (t.End.1), quando incubadas na presença de macrófagos tratados com CTX (0,3µg/mL) ou sobrenadantes desses macrófagos apresentaram significativa inibição da proliferação, da adesão aos seus ligantes naturais (colágeno I-10µg/mL; fibronectina-3µg/mL e laminina-10 µg/mL) e do número de células migradas, em modelo de Wound healing, bem como a velocidade de migração, avaliada em Time Lapse. Consequentemente, houve inibição da formação de tubos no matrigel-3D, acompanhada pela diminuição da concentração de VEGF e TNF-. Ainda, o bloqueio dos receptores peptídeo formil (FPRs) aboliu as atividades inibitórias dos macrófagos tratados com a toxina, evidenciando a importância destes receptores para a ação da CTX sobre a atividade antiangiogênica de macrófagos. / CTX stimulates, in vivo and in vitro, the macrophages secretory capacity in the presence of tumor, accompanied by a significant tumor mass reduction and tumor cells or inhibition of the proliferation, respectively. Considering the immunomodulatory action of CTX, the pivotal importance of macrophages in the genesis, tumor progression and in the stromal microenvironment, the macrophages secretory activity treated with CTX on angiogenesis was investigated in vitro. Thymic endothelial cells (t.End.1), when incubated in the presence of macrophages treated with CTX (0.3g/ml) or supernatants of these macrophages showed significant inhibition of proliferation, adhesion to its natural ligands (Type I collagen-10mg/ml ; fibronectin-3g/ml and laminin-10/mL), and of the cell number migrated in Wound healing model as well as the cell migration velocity, evaluated in Time Lapse. Consequently, there was inhibition of the cappilary-like tube formation on matrigel-3D matrix, accompanied by VEGF and TNF- secretion decrease. Further, the formyl peptide receptor (FPRs) blockade abolished the inhibitory activity of the macrophages treated with the toxin, suggesting the importance of these receptors to the action of CTX on the macrophages antiangiogenic activity.

O câncer é um problema de saúde pública mundial, apresentando acréscimo na sua incidência a cada ano. O seu processo de evolução ainda não foi completamente desvendado, dificultando a elaboração de terapias adequadas. Na busca por um melhor prognóstico, pesquisas recentes têm discutido o papel das citocinas inflamatórias, do nicho perivascular e das células-tronco nos mecanismos de desenvolvimento e manutenção dos tumores malignos. Os tumores de glândula salivar representam uma pequena porcentagem das patologias malignas da região de cabeça e pescoço, podendo ocorrer em adultos e em crianças. O diagnóstico dificilmente é precoce e a taxa de sobrevida é extremamente baixa comparada aos demais tumores da região. Assim, este estudo teve como objetivo estudar as células provenientes dos tumores de glândula salivar do tipo adenoide cístico (CAC) e adenocarcinoma NOS (AdNOS) quanto ao seu perfil imunofenotípico, quanto à existência ou não de células-tronco tumorais nessa população, bem como investigar possíveis modificações na ativação de STAT3, Akt e ERK (moléculas envolvidas em vias de sinalização de manutenção do tumor), quando em contato com fatores secretados por células endoteliais. Foram coletados 5 CACs e 4 AdNOS, no Hospital da Universidade de Michigan (Ann Arbor, MI, EUA), durante 2010 e 2012. As células foram isoladas e caracterizadas em citometria de fluxo em P0 e P7, demonstrando um perfil de células CD44+ALDH+Lin- variando de 0,33 a 3,19% e 0,36 a 2,00%, respectivamente, entre 5 linhagens avaliadas. Na avaliação por western blotting, a e-caderina, o Snail e a actina de músculo liso foram ausentes em todos os tipos tumorais, enquanto que a citoqueratina 20 (Ck20) foi presente apenas nos AdNOS. Comparando os tumores com suas metástases, a presença de Ck20, p63 e β-catenina foi semelhante, enquanto que citoqueratina 7, a vimentina e o Bmi-1 foram maiores nas metástases. Tanto os AdNOS quanto CACs apresentaram receptores para IL-6, IL-8 e EGF. Foi observado que mediadores solúveis liberados pelas células endoteliais foram capazes de fosforilar STAT3, Akt e ERK em todas as células salivares estudadas, no entanto, a proteína recombinante humana IL-6, isoladamente, não foi capaz de ativar Akt. Orosferas foram geradas em todos os tipos tumorais, demonstrando o potencial de auto-renovação celular. Um maior número de esferas foi observado nas células metastáticas em relação às primárias. Células CD44+ALDH+, comparadas com CD44-ALDH-, geraram mais esferas, quando plaqueadas em alta densidade (5.000 células). No entanto, o inverso foi encontrado, quando uma única célula foi utilizada para o ensaio (p>0,05). Devido à dificuldade de obtenção e manipulação de células de tumores de glândula salivar, ainda há muito que se investigar mecanisticamente. Considerando a fosforilação de STAT3 na presença de IL-6, semelhante ao verificado em outros tumores, o uso de anticorpos contra IL-6, talvez sejam uma opção no futuro. / Cancer is a public health problem worldwide, with an increase in incidence every year. The process of its evolution is still not completely understood, hindering the development of appropriate therapies. In the search for a better prognosis, recent reports have discussed the role of inflammatory cytokines, perivascular niche and stem cells in the mechanisms of development and maintenance of malignant tumors. The salivary gland tumors represent a small percentage of malignancies of the head and neck and can occur in both adults and children. Early diagnosis is difficult and the survival rate is extremely low compared to other tumors in the same region. Thus, this study aimed to study cells from the adenoid cystic carcinoma (ACC) and adenocarcinoma NOS (AdNOS) tumors of salivary gland regarding its immunophenotypic profile and the existence or absence of tumor stem cells in this population, as well as investigate possible changes in the activation of STAT3, Akt and ERK (molecules involved in signaling pathways of tumor maintenance), when exposed to factors secreted by endothelial cells. ACCs (n=5) and AdNOS (n=4) were collected at the Hospital of the University of Michigan (Ann Arbor, MI, USA), during 2010 to 2012. Cells were isolated and characterized by flow cytometry at P0 and P7, showing a profile of ALDH+CD44+Lin- ranging from 0.33% to 3.19% and 0.36% and 2.00%, respectively, between 5 cell lines evaluated. In the protein profile, e-cadherin, Snail and SMA were absent in all tumor types. Ck20 was present only in AdNOS. Comparing primary tumors and their metastases, the presence of Ck20, and p63 β-catenin was similar, while Ck7, vimentin and Bmi-1 were higher in metastases. Both AdNOS as ACCs had receptors for IL-6, IL-8 and EGF. It was observed that soluble mediators released by endothelial cells were able to activate STAT3, Akt and ERK phosphorylation in all cells studied. However, recombinant human IL-6 alone was not able to activate Akt. Orospheres were generated in all tumor types, indicating the potential for cellular self-renewal. Highest number of spheres was observed in metastatic cells compared to primary. ALDH+CD44+ cells compared to ALDH-CD44- generated more spheres when plated in high density (5,000 cells), however, the opposite was found when one single cell seed was evaluated (p> 0.05). There is doubt if these cell markers would be consider for a stem cell model in salivary tumors. Due to the difficulty of obtaining and manipulating salivary gland tumor cells, there is still much to investigate mechanistically. As the phosphorylation of STAT3, in the presence of IL-6, was similar to that observed in other tumors, the use of antibodies against IL-6, may be an option in the future.