THE ROLE OF THE VIRAL NEF PROTEIN AS A MEDIATOR OF HIV-1 INDUCED ENDOTHELIAL DYSFUNTION

poster abstractWith the prevalence of antiviral therapy in the developed world, many HIV-1-infected people die of diseases other than AIDS. One of the emerging major causes is cardiovascular disease, leading to the prediction that the majority of HIV-1 patients are expected to develop cardiovascular complications. Endothelial dysfunction is thought to be a key event in the development of cardiovascular diseases, particular-ly atherosclerosis. Assays testing the effect of HIV-1 on endothelial ac-tivation shows that direct contact with HIV-1 infected T cells enhance endothelial cell activation to a greater extent than HIV-1 alone, sug-gesting an intracellular HIV-1 protein is responsible for endothelial ac-tivation. The HIV-1 viral protein Nef, which is responsible for T cell ac-tivation and maintenance of high viral loads in vivo, has been shown to mediate its own transfer to bystander cells. We demonstrate here for the first time that Nef induces nanotube-like conduits connecting T cells and endothelial cells. We also show that Nef is transferred from T cells to endothelial cells via these nanotubes, and is necessary and sufficient for endothelial cell activation. Moreover, we show that SIV-infected macaques exhibit endothelial Nef expression in coronary ar-teries. Nef expression in endothelial cells causes endothelial apoptosis, ROS and MCP-1 production. Interestingly, a Nef SH3 binding site mu-tant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through an NADPH oxidase- and ROS-dependent mechanism, while Nef-induced MCP-1 production is NF-kB dependent. Taken together, these data suggest that Nef can mediate its transfer from T cells to endothelial cells through nanotubes to en-hance endothelial dysfunction in vivo. Thus, Nef is a promising new therapeutic target for reducing the risk for cardiovascular disease in the HIV-1 positive population

Untersuchungen zur Funktion endothelialer Transkriptionsfaktoren bei der Angiogenese

Mit Hilfe des Cre-loxP Rekombinationssystems können Gene zelltypspezifisch inaktiviert werden. Hier wurden die genregulatorischen Elemente des flk-1 Gens mit dem Cre Rekombinase Gen fusioniert und transgene Mauslinien etabliert (flk-1-Cre). Diese Mäuse wurden mit der Reportermauslinie Rosa26R gekreuzt, so dass in doppelt transgenen Nachkommen die Cre Aktivität durch LacZ-Färbung analysiert werden konnte. Die Untersuchung von flk-1-Cre/ Rosa26R Embryonen und adulten Tieren zeigte eine spezifische Färbung der Endothelzellen von Blutgefäßen in fast allen Organsystemen. Außerdem wurde die Cre Expression in Gefäßen von experimentellen BFS-1 Tumoren nachgewiesen. Flk-1-Cre Mauslinien können in Zukunft verwendet werden, um gefloxte Zielgene spezifisch in Endothelzellen zu inaktivieren. Die Rolle von Hypoxie-induzierbaren Faktoren (HIF) in der embryonalen Gefäßentwicklung wurde untersucht, indem HIF Signalwege im Endothel blockiert wurden. Dazu wurde eine dominant negative HIF2-alpha Mutante verwendet (HIFdn), die mittels der regulatorischen Elemente des flk-1 Gens spezifisch in Endothelzellen von transgenen Mausembryonen exprimiert wurde. Die HIFdn transgenen Embryonen zeigten eine Wachstumsretardierung und starben an Tag 11,5 der Embryonalentwicklung. In den transgenen Embryonen wurden primitive Gefäßstrukturen gebildet, was auf eine normale Vaskulogenese hinwies. Diese unreifen Gefäße wurden aber nicht remodelliert, da keine sprossende Angiogenese auftrat. In Herzen von HIFdn transgenen Embryonen zeigten sich Defekte in der Bildung von Trabekeln und in der Ausbildung der Herzschleife. Die Analyse der Genexpression zeigte, dass endotheliale Gene wie tie-2, flt-1 und flk-1 in transgenen Embryonen stark reduziert waren. Diese Ergebnisse lassen den Schluss zu, dass endotheliale HIF eine essentielle Rolle in der embryonalen Angiogenese spielen. Die Regulation des humanen VEGF Rezeptor-2 Gens (KDR) wurde untersucht. Es konnte gezeigt werden, dass der Transkriptionsfaktor HIF2-alpha, nicht aber der verwandte HIF1-alpha die Aktivität des KDR Promotors in Reportergenexperimenten stimulierte. Potentielle HIF2 Bindungsstellen im KDR Promotor wurden identifiziert. Durch die Mutation einer dieser Bindungsstellen wurde die Aktivierbarkeit des Promotors stark reduziert. Im Vergleich zum Mausgen, zeigte das 1. Intron des humanen KDR Gens eine Region großer Sequenzidentität. Im Mausgen enthält diese Region („Enhancer“) Bindungsstellen für Transkriptionsfaktoren und sie ist essentiell für die endothelspezifische Expression in vivo. Die Bindungsstellen für diese Transkriptionsfaktoren (SCL/Tal-1, Gata, Ets) sind im 1. Intron des humanen Gens konserviert, so dass sie wahrscheinlich an der Regulation der Genexpression beteiligt sind

Extracellular vesicles: Novel communicators in lung diseases

This work is licensed under a Creative Commons Attribution 4.0 International License.The lung is the organ with the highest vascular density in the human body. It is therefore perceivable that the endothelium of the lung contributes significantly to the circulation of extracellular vesicles (EVs), which include exosomes, microvesicles, and apoptotic bodies. In addition to the endothelium, EVs may arise from alveolar macrophages, fibroblasts and epithelial cells. Because EVs harbor cargo molecules, such as miRNA, mRNA, and proteins, these intercellular communicators provide important insight into the health and disease condition of donor cells and may serve as useful biomarkers of lung disease processes. This comprehensive review focuses on what is currently known about the role of EVs as markers and mediators of lung pathologies including COPD, pulmonary hypertension, asthma, lung cancer and ALI/ARDS. We also explore the role EVs can potentially serve as therapeutics for these lung diseases when released from healthy progenitor cells, such as mesenchymal stem cells.NiH R01DA042715NIH R01HL129875NIH P20 GM10363

Increased cardiovascular disease risk in the HIV-positive population on ART: potential role of HIV-Nef and Tat

With effective antiretroviral therapy (ART), many HIV-infected people die of diseases other than acquired immune deficiency syndrome (AIDS). In particular, coronary artery disease has emerged as one of most critical complications of HIV infection and a major cause of morbidity and mortality. Although reportedly antiretroviral combination therapy itself may accelerate atherosclerosis by enhancing dyslipidemia, most recent epidemiological studies support the notion that HIV infection itself contributes to cardiovascular disease. However, it is still a mystery how the virus can contribute to cardiovascular disease development even while suppressed by ARTs. This review discusses the current understanding of interactions between HIV infection and cardiovascular diseases in both clinical and experimental studies with special focus on those viral proteins that are still produced by HIV. This will help infectious disease/vascular biology experts to gain insights into the pathophysiological mechanisms of HIV-associated cardiovascular disease and new trends to treat and prevent cardiovascular disease in the HIV-infected population

The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor

AbstractThe 60 kDa tumor necrosis factor receptor (TNFR60) is regarded as the major signal transducer of TNF-induced cellular responses, whereas the signal capacity and role of the 80 kDa TNFR (TNFR80) remain largely undefined. We show here that the transmembrane form of TNF is superior to soluble TNF in activating TNFR80 in various systems such as T cell activation, thymocyte proliferation, and granulocyte/macrophage colony-stimulating factor production. Intriguingly, activation of TNFR80 by membrane TNF can lead to qualitatively different TNF responses such as rendering resistant tumor cells sensitive to TNF-mediated cytotoxicity. This study demonstrates that the diversity of TNF effects can be controlled through the differential sensitivity of TNFR80 for the two forms of TNF and suggests an important physiological role for TNFR80 in local inflammatory responses

Long-term survival and radiological results of the Duracon™ total knee arthroplasty

Purpose: The aim of this study was to analyse the long-term (>tenyears) survival rate and radiological results of the Duracon TKA. Methods: Between 1992 and 1999 159 Duracon TKA were implanted at our institution. A Kaplan-Meier survival analysis for the endpoints exchange, addition or removal of any component for any reason, revision due to aseptic loosening and mechanical failure was performed. Radiological long-term (>tenyears) follow-up (FU) analysis was performed according to the Knee Society Radiographic Evaluation and Scoring System. Results: Mean age at surgery was 74.3years, 28% were male, and 89% had primary osteoarthritis as diagnosis. Mean FU for survival analysis was 10.9years (SD 4.2). A total of 58% of the patients died during follow-up. Three patients (2.1%) were lost to follow-up and five TKA (3.1%) were revised. After tenyears the mean survival was 97.7%, 99.4% and 98.3% for the aforementioned endpoints, respectively. Mean radiological FU was 11.8years (SD 2.3). We found no significant change in alignment of the components or axis over time. Progressive radiolucencies were found in nine TKA (17%), mainly around the tibial component (95%). Conclusion: The Duracon TKA showed excellent long-term survival comparable to data from national registers and to other successful designs. Radiological changes found on plain radiographs were scarce after almost 12years of radiological follow-up indicating good implant stability

Endogenous Transmembrane TNF-Alpha Protects Against Premature Senescence in Endothelial Colony Forming Cells

RATIONALE: Transmembrane tumor necrosis factor-α (tmTNF-α) is the prime ligand for TNF receptor 2, which has been shown to mediate angiogenic and blood vessel repair activities in mice. We have previously reported that the angiogenic potential of highly proliferative endothelial colony-forming cells (ECFCs) can be explained by the absence of senescent cells, which in mature endothelial cells occupy >30% of the population, and that exposure to a chronic inflammatory environment induced premature, telomere-independent senescence in ECFCs. OBJECTIVE: The goal of this study was to determine the role of tmTNF-α in the proliferation of ECFCs. METHODS AND RESULTS: Here, we show that tmTNF-α expression on ECFCs selects for higher proliferative potential and when removed from the cell surface promotes ECFC senescence. Moreover, the induction of premature senescence by chronic inflammatory conditions is blocked by inhibition of tmTNF-α cleavage. Indeed, the mechanism of chronic inflammation-induced premature senescence involves an abrogation of tmTNF/TNF receptor 2 signaling. This process is mediated by activation of the tmTNF cleavage metalloprotease TNF-α-converting enzyme via p38 MAP kinase activation and its concurrent export to the cell surface by means of increased iRhom2 expression. CONCLUSIONS: Thus, we conclude that tmTNF-α on the surface of highly proliferative ECFCs plays an important role in the regulation of their proliferative capacity