Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

mTORC2 is an important signaling intermediary in VEGF-mediated endothelial cell proliferation, survival and migration

Objective: The vascular endothelial growth factor (VEGF) is a prominent¦contributor of tumor angiogenesis. VEGF induces endothelial cell migration,¦proliferation and survival, which are critical steps for the development of new¦blood vessels, through the activation of the Mek/Erk and PI3K/Akt signaling¦pathways. Recent findings have demonstrated that mTORC2 regulates Akt and¦Erk in endothelial cells. The role of mTORC2 in VEGF-mediated endothelial¦cell responses has however not been characterized.¦Methods: We used human umbilical vein endothelial cells (HUVEC). The¦effects of VEGF on the Mek/Erk and PI3K/Akt pathway were analyzed by¦Western blot. Inhibition of mTORC2 was achieved using small interfering¦RNAs to rictor. Cell proliferation rate was assessed by BrdU incorporation and¦immunocytofluorescence. Apoptosis rate was determined by ELISA as well as¦propidium iodine staining and FACS analysis. Migration of endothelial cells¦was evaluated using a modified Boyden chamber assay.¦Results:Wefound thatVEGF activatesmTORC2 in endothelial cells. Indeed,¦treatment of endothelial cells with VEGF increases Akt phosphorylation, a¦downstream effector of mTORC2. We have further determined the role¦of mTORC2 in VEGF signaling by knocking down rictor, a component¦of mTORC2. We observed that VEGF failed to activate Akt and Erk in¦endothelial cells transfected with rictor siRNA. To next analyze the functional¦significance of mTORC2 inhibition on VEGF-mediated endothelial cell¦responses we performed proliferation, survival and migration assays. We found¦that VEGF failed to induce endothelial cell proliferation, survival and migration¦in endothelial cell lacking mTORC2 activity.¦Conclusion: These results show that mTORC2 is an important signaling¦intermediary in VEGF-induced endothelial cell responses and thus represents¦an interesting target to block VEGF-induced angiogenesis.

Mechanisms of interaction of therapeutic nanoparticles with cells

Nanoparticles (NPs) have gained a lot of interest in recent years due to their huge potential for applications in industry and medicine. Their unique properties offer a large number of attractive possibilities in the biomedical field, providing innovative tools for diagnosis of diseases and for novel therapies. Nevertheless, a deep understanding of their interactions with living tissues and the knowledge about their possible effects in the human body are necessary for the safe use of nanoparticulate formulations. The aim of this PhD project was to study in detail the interactions of therapeutic NPs with living cells, including cellular uptake and release, cellular localization and transport across the cell layers. Moreover, the effects of NPs on the cellular metabolic processes were determined using adapted in vitro assays. We evaluated the biological effect of several NPs potentially used in the biomedical field, including titanium dioxide (Ti02) NPs, 2-sized fluorescent silica NPs, ultrasmall superparamagnetic iron oxide (USPIO) NPs, either uncoated or coated with oleic acid or with polyvinylamine (aminoPVA) and poly(lactic-co-glycolic acid) - polyethylene-oxide (PLGA-PEO) NPs. We have found that the NPs were internalized by the cells, depending on their size, chemical composition, surface coating and also depending on the cell line considered. The uptake of aminoPVA-coated USPIO NPs by endothelial cells was enhanced in the presence of an external magnetic field. None of the tested USPIO NPs and silica NPs was transported across confluent kidney cell layers or brain endothelial cell layers, even in the presence of a magnetic field. However, in an original endothelium-glioblastoma barrier model which was developed, uncoated USPIO NPs were directly transferred from endothelial cells to glioblastoma cells. Following uptake, Ti02 NPs and uncoated USPIO NPs were released by the kidney cells, but not by the endothelial cells. Furthermore, these NPs induced an oxidative stress and autophagy in brain endothelial cells, possibly associated with their enhanced agglomeration in cell medium. A significant DNA damage was found in brain endothelial cells after their exposure to TiO2NPs. Altogether these results extend the existing knowledge about the effects of NPs on living cells with regard to their physicochemical characteristics and provide interesting tools for further investigation. The development of the in vitro toxicological assays with a special consideration for risk evaluation aims to reduce the use of animal experiments. -Les nanoparticules (NPs) présentent beaucoup d'intérêt dans le domaine biomédical et industriel. Leurs propriétés uniques offrent un grand nombre de possibilités de solutions innovantes pour le diagnostique et la thérapie. Cependant, pour un usage sûr des NPs il est nécessaire d'acquérir une connaissance approfondie des mécanismes d'interactions des NPs avec les tissus vivants et de leur effets sur le corps humain. Le but de ce projet de thèse était d'étudier en détail les mécanismes d'interactions de NPs thérapeutiques avec des cellules vivantes, en particulier les mécanismes d'internalisation cellulaire et leur subséquente sécrétion par les cellules, leur localisation cellulaire, leur transport à travers des couches cellulaires, et l'évaluation des effets de NPs sur le métabolisme cellulaire, en adaptant les méthodes existante d'évaluation cyto-toxico logique s in vitro. Pour ces expériences, les effets biologiques de nanoparticules d'intérêt thérapeutique, telles que des NPs d'oxyde de titane (TiO2), des NPs fluorescents de silicate de 2 tailles différentes, des NPs, d'oxyde de fer super-para-magnétiques ultra-petites (USPIO), soit non- enrobées soit enrobées d'acide oléique ou de polyvinylamine (aminoPVA), et des NPs d'acide poly(lactique-co-glycolique)-polyethylene-oxide (PLGA-PEO) ont été évalués. Les résultats ont démontré que les NPs sont internalisées par les cellules en fonction de leur taille, composition chimique, enrobage de surface, et également du type de cellules utilisées. L'internalisation cellulaire des USPIO NPs a été augmentée en présence d'un aimant externe. Aucune des NPs de fer et de silicate n'a été transportée à travers des couches de cellules épithéliales du rein ou endothéliales du cerveau, même en présence d'un aimant. Cependant, en développant un modèle original de barrière endothélium-glioblastome, un transfert direct de NPs d'oxyde de fer de cellule endothéliale à cellule de glioblastome a été démontré. A la suite de leur internalisation les NPs d'oxyde de fer et de titane sont relâchées par des cellules épithéliales du rein, mais pas des cellules endothéliales du cerveau. Dans les cellules endothéliales du cerveau ces NPs induisent en fonction de leur état d'agglomération un stress oxydatif et des mécanismes d'autophagie, ainsi que des dommages à l'ADN des cellules exposées aux NPs d'oxyde de titane. En conclusion, les résultats obtenus élargissent les connaissances sur les effets exercés par des NPs sur des cellules vivantes et ont permis de développer les outils expérimentaux pour étudier ces effets in vitro, réduisant ainsi le recours à des expériences sur animaux.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Emerging paradigms and questions on pro-angiogenic bone marrow-derived myelomonocytic cells.

Cancer-related inflammation has emerged in recent years as a major event contributing to tumor angiogenesis, tumor progression and metastasis formation. Bone marrow-derived and inflammatory cells promote tumor angiogenesis by providing endothelial progenitor cells that differentiate into mature endothelial cells, and by secreting pro-angiogenic factors and remodeling the extracellular matrix to stimulate angiogenesis though paracrine mechanisms. Several bone marrow-derived myelonomocytic cells, including monocytes and macrophages, have been identified and characterized by several laboratories in recent years. While the central role of these cells in promoting tumor angiogenesis, tumor progression and metastasis is nowadays well established, many questions remain open and new ones are emerging. These include the relationship between their phenotype and function, the mechanisms of pro-angiogenic programming, their contribution to resistance to anti-angiogenic treatments and to metastasis and their potential clinical use as biomarkers of angiogenesis and anti-angiogenic therapies. Here, we will review phenotypical and functional aspects of bone marrow-derived myelonomocytic cells and discuss some of the current outstanding questions.

Between Immunology And Tolerance: Controlling Immune Responses Employing Tolerogenic Dendritic Cells

Dendritic cells (DCs) are the most efficient antigen presenting cells, they provide co-stimulation, are able to secrete various proinflammatory cytokines and therefore play a pivotal role in shaping adaptive immune responses. Moreover, they are important for the promotion and maintenance of central and peripheral tolerance through several mechanisms like the induction of anergy or apoptosis in effector T cells or by promoting regulatory T cells. The murine CD8α+ (MuTu) dendritic cell line was previously derived and described in our laboratory. The MuTu cell line has been shown to maintain phenotypical and functional characteristics of endogenous CD8α+ DCs. They are able to cross-present exogenous antigens to CD8+ T cells and produce interleukin (IL-) 12 upon engagement of Toll like receptors. The cell line constitutes an infinite source of homogenous, phenotypically well-defined dendritic cells. This allows us to investigate the role and potential of specific molecules in the induction as well as regulation of immune responses by DCs in a rational and standardized way. In a first project the MuTu dendritic cell line was transduced in order to stably express the immunosuppressive molecules IL-10, IL-35 or the active form of TGF-β (termed IL-10+DC, IL-35+DC or actTGFβ+DC). We investigated the capability of these potentially suppressive or tolerogenic dendritic cell lines to induce immune tolerance and explore the mechanisms behind tolerance induction. The expression of TGF-β by the DC line did not affect the phenotype of the DCs itself. In contrast, IL-10+ and IL-35+DCs were found to exhibit lower expression of co-stimulatory molecules and MHC class I and II, as well as reduced secretion of pro-inflammatory cytokines upon activation. In vitro co-culture with IL-35+, IL10+ or active TGFβ+ DCs interfered with function and proliferation of CD4+ and CD8+ T cells. Furthermore, IL-35 and active TGF-β expressing DC lines induced regulatory phenotype on CD4+ T cells in vitro without or with expression of Foxp3, respectively. In different murine cancer models, vaccination with IL-35 or active TGF-β expressing DCs resulted in faster tumor growth. Interestingly, accelerated tumor growth could be observed when IL-35-expressing DCs were injected into T cell-deficient RAG-/- mice. IL-10expressing DCs however, were found to rather delay tumor growth. Besides the mentioned autocrine effects of IL-35 expression on the DC line itself, we surprisingly observed that the expression of IL-35 or the addition of IL-35 containing medium enhances neutrophil survival and induces proliferation of endothelial cells. Our findings indicate that the cytokine IL-35 might not only be a potent regulator of adaptive immune responses, but it also implies IL-35 to mediate diverse effects on an array of cellular targets. This abilities make IL-35 a promising target molecule not only for the treatment of auto-inflammatory disease but also to improve anti-cancer immunotherapies. Indeed, by applying active TGFβ+ in murine autoimmune encephalitis we were able to completely inhibit the development of the disease, whereas IL-35+DCs reduced disease incidence and severity. Furthermore, the preventive transfer of IL-35+DCs delayed rejection of transplanted skin to the same extend as the combination of IL-10/actTGF-β expressing DCs. Thus, the expression of a single tolerogenic molecule can be sufficient to interfere with the adequate activation and function of dendritic cells and of co-cultured T lymphocytes. The respective mechanisms of tolerance induction seem to be different for each of the investigated molecule. The application of a combination of multiple tolerogenic molecules might therefore evoke synergistic effects in order to overcome (auto-) immunity. In a second project we tried to improve the immunogenicity of dendritic cell-based cancer vaccines using two different approaches. First, the C57BL/6 derived MuTu dendritic cell line was genetically modified in order to express the MHC class I molecule H-2Kd. We hypothesized that the expression of BALB/c specific MHC class I haplotype (H-2Kd) should allow the priming of tumor-specific CD8+ T cells by the otherwise allogeneic dendritic cells. At the same time, the transfer of these H-2Kd+ DCs into BALB/c mice was thought to evoke a strong inflammatory environment that might act as an "adjuvant", helping to overcome tumor induced immune suppression. Using this so called "semi-allogeneic" vaccination approach, we could demonstrate that the delivery of tumor lysate pulsed H-2Kd+ DCs significantly delayed tumor growth when compared to autologous or allogeneic vaccination. However, we were not able to coherently elucidate the cellular mechanisms underlying the observed effect. Second, we generated MuTu DC lines which stably express the pro-inflammatory cytokines IL-2, IL-12 or IL-15. We investigated whether the combination of DC vaccination and local delivery of pro-inflammatory cytokines might enhance tumor specific T cell responses. Indeed, we observed an enhanced T cell proliferation and activation when they were cocultured in vitro with IL-12 or IL-2-expressing DCs. But unfortunately we could not observe a beneficial or even synergistic impact on tumor development when cytokine delivery was combined with semi-allogeneic DC vaccination.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Endothelial, but not smooth muscle, peroxisome proliferator-activated receptor β/δ regulates vascular permeability and anaphylaxis.

BACKGROUND: Remodeling of quiescent vessels with increases in permeability, vasodilatation, and edema are hallmarks of inflammatory disorders. Factors involved in this type of remodeling represent potential therapeutic targets. OBJECTIVES: We investigated whether the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) β/δ, a regulator of metabolism, fibrosis, and skin homeostasis, is involved in regulation of this type of remodeling. METHODS: Wild-type and various Pparb/d mutant mice were used to monitor dermal acute vascular hyperpermeability (AVH) and passive systemic anaphylaxis-induced hypothermia and edema. PPARβ/δ-dependent kinase activation and remodeling of endothelial cell-cell junctions were addressed by using human endothelial cells. RESULTS: AVH and dilatation of dermal microvessels stimulated by vascular endothelial growth factor A, histamine, and thrombin are severely compromised in PPARβ/δ-deficient mice. Selective deletion of the Pparb/d-encoding gene in endothelial cells in vivo similarly limits dermal AVH and vasodilatation, providing evidence that endothelial PPARβ/δ is the major player in regulating acute dermal microvessel remodeling. Furthermore, endothelial PPARβ/δ regulatory functions are not restricted to the skin vasculature because its deletion in the endothelium, but not in smooth muscle cells, also leads to reduced systemic anaphylaxis, the most severe form of allergic reaction, in which an acute vascular response plays a key role. PPARβ/δ-dependent AVH activation likely involves the activation of mitogen-activated protein kinase and Akt pathways and leads to downstream destabilization of endothelial cell-cell junctions. CONCLUSION: These results unveil not only a novel function of PPARβ/δ as a direct regulator of acute vessel permeability and dilatation but also provide evidence that antagonizing PPARβ/δ represents an important strategy to consider for moderating diseases with altered endothelial integrity, such as acute inflammatory and allergic disorders.

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

BACKGROUND: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. RESULTS: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. CONCLUSIONS: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.

Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.

Targeting and translocation of endothelial nitric oxide synthase

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca2+/calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co- and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca2+. These rapid receptor-mediated effects are seen not only for "classic" eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Coronary endothelial dysfunction after ischemia and reperfusion: a new therapeutic target?

Although cardiac ischemia is usually characterized as a disease of the myocyte, it is clear that the vasculature, and especially endothelial cells, is also a major target of this pathology. Indeed, using a rat model of ischemia/reperfusion, we were able to detect severe endothelial dysfunction (assessed as a decreased response to acetylcholine) after acute or chronic reperfusion. Given the essential role of the endothelium in the regulation of vascular tone, as well as platelet and leukocyte function, such a severe dysfunction could lead to an increased risk of vasospasm, thrombosis and accelerated atherosclerosis. This dysfunction can be prevented by free radical scavengers and by exogenous nitric oxide. Endothelial dysfunction can also be prevented by preconditioning with brief periods of intermittent ischemia, thus extending to coronary endothelial cells the concept of endogenous protection previously described at the myocyte level. Experiments performed on cultured cells showed that the endothelial protection induced by free radical scavengers or by preconditioning was due to a lesser expression of endothelial adhesion molecules such as intercellular adhesion molecule-1, leading to a lesser adhesion of neutrophils to endothelial cells. Identification of the mechanisms of this protection may lead to the development of new strategies aimed at protecting the vasculature in ischemic heart diseases.

Wine, alcohol and atherosclerosis: clinical evidences and mechanisms

Atherosclerosis is a chronic inflammatory disease which may cause obstructions of the coronary, cerebral and peripheral arteries. It is typically multifactorial, most often dependent on risk factors such as hypercholesterolemia, diabetes, smoking, hypertension, sedentarism, and obesity. It is the single main cause of death in most developed countries due to myocardial infarction, angina, sudden death, and heart failure. Several epidemiological studies suggest that moderate alcohol intake, especially red wine, decrease cardiac mortality due to atherosclerosis. The alcohol effect is described by a J curve, suggesting that moderate drinkers may benefit while abstainers and heavy drinkers are at higher risk. Experimental studies indicate that most beneficial effects of drinking are attributable to flavonoids that are present in red wine, purple grape juice and several fruits and vegetables. The mechanisms include antiplatelet actions, increases in high-density lipoprotein, antioxidation, reduced endothelin-1 production, and increased endothelial nitric oxide synthase expression which causes augmented nitric oxide production by endothelial cells. These findings lead to the concept that moderate red wine drinking, in the absence of contraindications, may be beneficial to patients who are at risk of atherosclerotic cardiovascular events. Moreover, a diet based on fruits and vegetables containing flavonoids may be even more beneficial.