Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract.

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the "HSP60 pathway." It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the "auto-immune hypothesis of atherosclerosis."

Methodological obstacles in knocking down small noncoding RNAs.

In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed "orphan snoRNAs," lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A(391) within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.

p16INK4A is a robust in vivo biomarker of cellular aging in human skin.

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury.

BACKGROUND: Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration. METHODS: Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10(6) cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals. RESULTS: We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats. CONCLUSION: Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflammatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.

Interaction of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro.

This study examined the direct interaction of serotonin (5-hydroxytryptamine (5-HT)) with Aspergillus species. Accumulation of 5-HT in aspergilli was investigated by immunofluorescence staining and laser confocal scanning microscopy. The influence of 5-HT on fungal ergosterol content, cell membrane integrity, fungal growth and hyphal elongation was determined. 5-HT was localised in the cytoplasm of Aspergillus spp., as 5-HT fluorescent signals appeared after 30min at 4 degrees C and in the presence of inhibitors of oxidative phosphorylation. 5-HT treatment of Aspergillus spp. significantly affected ergosterol synthesis, fungal cell membrane integrity and hyphal elongation (P<0.05). 5-HT treatment for 4h resulted in a lag of re-growth (post-antifungal effect). In conclusion, our findings suggest that 5-HT affects hyphal growth and diminishes fungal cell membrane integrity.

Atherosclerosis research from past to present--on the track of two pathologists with opposing views, Carl von Rokitansky and Rudolf Virchow.

It is now clear that inflammation plays a key role in atherogenesis. As a matter of fact, signs of inflammation of atherosclerotic plaques have been observed for centuries and also constituted the basis for a fierce controversy in the 19th century between the prominent Austrian pathologist Carl von Rokitansky and his German counterpart, Rudolf Virchow. While the former attributed a secondary role to these inflammatory arterial changes, Virchow considered them to be of primary importance. We had the unique opportunity to address this controversy by investigating atherosclerotic specimens from autopsies performed by Carl von Rokitansky up to 178 years ago. Twelve atherosclerotic arteries originally collected between the years 1827 to 1885 were selected from the Collection Rokitansky of the Federal Museum of Pathological Anatomy, Vienna Medical University. Using modern sophisticated immunohistochemical and immunofluorescence techniques, it was shown that various cellular intralesional components, as well as extracellular matrix proteins, were preserved in the historic atherosclerotic specimens. Most importantly, CD3 positive cells were abundant in early lesions, thus, rather supporting Virchows's view, that inflammation is an initiating factor in atherogenesis. Furthermore, we hope to have opened a new and intriguing possibility to study various pathological conditions using valuable historical specimens.

Differential regulation of apoptotic cell death in senescent human cells.

Aging of human cells can be reproduced in monolayer cultures, revealing the phenotype of replicative senescence. It was shown that diploid human fibroblasts enter a stable growth arrest phenotype at the end of their lifespan and, in particular, these cells are resistant to various apoptotic stimuli. In contrast, human endothelial cells from the umbilical vein (HUVEC) acquire a proapoptotic phenotype when reaching senescence and this probably results from reactive oxygen species (ROS) induced damage and associated signaling. Ceramides were shown to accumulate in senescent fibroblasts and are also known as potent regulators of apoptotic cell death. To further study age-associated changes in proneness to apoptosis between fibroblasts and endothelial cells, both cell types were challenged by administration of exogenous ceramide and apoptotic cell death was determined. While ceramide can efficiently induce apoptosis in both young and senescent cells of either histotype, quantitative evaluation of the data show that senescent fibroblasts are more resistant to apoptosis induction when compared to their young counterparts, whereas in the case of endothelial cells proneness for apoptosis is increased in senescent cells. Together, these data suggest significant differences in the regulation of apoptosis associated with senescence in fibroblasts and endothelial cells.

CenH3/CID incorporation is not dependent on the chromatin assembly factor CHD1 in Drosophila.

CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3(CENP-A), which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3(CID) in Drosophila. In contrast to the findings in fission yeast and vertebrate cells, our evidence clearly argues against such a role for CHD1 in Drosophila. CHD1 does not localize to centromeres in either S2 cells or developing fly embryos. Down-regulation of CHD1 in S2 cells by RNAi reveals unchanged levels of CenH3(CID) at the centromeres. Most notably, ablation of functional CHD1 in Chd1 mutant fly embryos does not interfere with centromere and kinetochore assembly, as the levels and localization of CenH3(CID), CENP-C and BubR1 in the mutant embryos remain similar to those seen in wild-type embryos. These results indicate that Drosophila CHD1 has no direct function in the incorporation of the centromeric H3 variant CenH3(CID) into chromatin. Therefore, centromeric chromatin assembly may involve different mechanisms in different organisms.

Autocrine formation of hepcidin induces iron retention in human monocytes.

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

Visfatin, an adipocytokine with proinflammatory and immunomodulating properties.

Adipocytokines are mainly adipocyte-derived cytokines regulating metabolism and as such are key regulators of insulin resistance. Some adipocytokines such as adiponectin and leptin affect immune and inflammatory functions. Visfatin (pre-B cell colony-enhancing factor) has recently been identified as a new adipocytokine affecting insulin resistance by binding to the insulin receptor. In this study, we show that recombinant visfatin activates human leukocytes and induces cytokine production. In CD14(+) monocytes, visfatin induces the production of IL-1beta, TNF-alpha, and especially IL-6. Moreover, it increases the surface expression of costimulatory molecules CD54, CD40, and CD80. Visfatin-stimulated monocytes show augmented FITC-dextran uptake and an enhanced capacity to induce alloproliferative responses in human lymphocytes. Visfatin-induced effects involve p38 as well as MEK1 pathways as determined by inhibition with MAPK inhibitors and we observed activation of NF-kappaB. In vivo, visfatin induces circulating IL-6 in BALB/c mice. In patients with inflammatory bowel disease, plasma levels of visfatin are elevated and its mRNA expression is significantly increased in colonic tissue of Crohn's and ulcerative colitis patients compared with healthy controls. Macrophages, dendritic cells, and colonic epithelial cells might be additional sources of visfatin as determined by confocal microscopy. Visfatin can be considered a new proinflammatory adipocytokine.

Dendritic cells regulate T-cell deattachment through the integrin-interacting protein CYTIP.

When T cells are primed by dendritic cells (DCs) to initiate antigen-specific immune responses screening for matching antigen receptor-MHC/peptide pairs takes place in DC-T-cell conjugates. For an immune response DC-T-cell conjugates formed during priming events need to dissolve. Although detailed knowledge on molecules involved in the conjugate formation is available, dissolving of them has not been considered to be an active process. Here, we identify CYTIP (cytohesin-interacting protein) to mediate DC-T-cell deattachment. CYTIP, which is induced during maturation of DCs, shortly accumulates to the contact zones with T cells within the first hour of coculture. Specific silencing of CYTIP results in stronger adhesion of DCs to T cells and to fibronectin. When a need for deattachment is created in a T-cell priming assay by only partially loading DCs with antigen, CYTIP silencing causes reduced priming capacity. Thus, CYTIP allows DCs to actively control DC-T-cell interactions.

BH3-only proteins Puma and Bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo.

Numerous p53 target genes have been implicated in DNA damage-induced apoptosis signaling, but proapoptotic Bcl-2 (B-cell leukemia 2) family members of the BH3 (Bcl-2 homolog region [BH] 3)-only subgroup appear to play the critical initiating role. In various types of cultured cells, 3 BH3-only proteins, namely Puma (p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting mediator of cell death), have been shown to initiate p53-dependent as well as p53-independent apoptosis in response to DNA damage and treatment with anticancer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids. To assess the in vivo relevance of these findings, we subjected mice lacking Puma, Noxa, or Bim to whole-body gamma-radiation or the glucocorticoid dexamethasone and compared lymphocyte survival with that in wild-type and BCL2-transgenic mice. Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of lymphocytes from the effects of gamma-radiation in vivo, and loss of Bim provided lower but significant protection in most lymphocytes, whereas Noxa deficiency had no impact. Furthermore, both Puma and Bim were found to contribute significantly to glucocorticoid-induced killing. Our results thus establish that Puma and Bim are key initiators of gamma-radiation- and glucocorticoid-induced apoptosis in lymphoid cells in vivo.

In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay.

OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti-endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis-inducing effect of AECAs in vivo. METHODS: The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD-200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA-positive serum samples from UCD-200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1-week-old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL. RESULTS: Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA-positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls. CONCLUSION: This study is the first to demonstrate the in vivo apoptosis-inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

Cellular and molecular composition of fibrous capsules formed around silicone breast implants with special focus on local immune reactions.

During the past 30 years, much debate has centered around side effects of silicone breast implants. Meta-analyses rejected the presumed relationship between silicone breast implants and connective tissues diseases but, in seeming contradiction, case reports about connective tissue diseases and rheumatoid symptoms continue to be published. We analyzed the cellular and molecular composition of fibrous capsules removed from patients at various times after surgery for diagnostic purposes (breast cancer relapse) or to relieve painful constrictive fibrosis. Frozen sections of capsule tissue were immunohistochemically stained for subsets of lymphocytes, macrophages, dendritic cells, fibroblasts, smooth muscle cells, for collagenous and non-collagenous extracellular matrix proteins, for heat shock protein 60 (HSP60) and for adhesion molecules. Massive deposition of fibronectin and tenascin was observed adjacent to the implant surface. The capsule/silicone implant contact zone was consistently characterized by a palisade-like single or multilayered cell accumulation consisting of HSP60+ macrophages and HSP60+ fibroblasts. Mononuclear cell infiltrates consisting of activated CD4+ T-cells, expressing CD25 and CD45RO, as well as macrophages were detected beneath the contact zone as well as perivascularly. Importantly, many Langerhans-cell like dendritic cells (DCs) were found with a predilection at the frontier layer zone abutting the silicone implant. Also, at this site, massive expression of ICAM-1, but not VCAM-1 or ELAM-1 emerged. Endothelial cells of the intracapsular neovasculature were P-Selectin+. Our results show that silicone induces a strong local T-cell immune response and future studies will determine the specificity and function of these T-lymphocytes.

Cross-linking of CD32 induces maturation of human monocyte-derived dendritic cells via NF-kappa B signaling pathway.

Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcgammaR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-kappaB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses.