(B) Time program of visfatin induction by IL-1 (10 ng/mL) over 48 h. Visfatin content material was also decided in HUVEC challenged for 18 h with either (C) TNF- (one to 10 ng/mL) or (D) Ang II (10 pmol/L to 1 ol/L). Info are the MEDChem Express 1802326-66-4meanEM of five unbiased experiments. P<0.05 vs nonstimulated cells. Representative gels are shown on the top.Using Western blot techniques, immunoreactive visfatin was detected in HUVEC cultures under non-inflammatory conditions (Figure 1A). However, after challenging the cells with a wellknown pro-inflammatory stimulus, such as the cytokine IL-1 (1 to 10 ng/mL 18 h), a concentration-dependent increase in the cellular levels of visfatin was observed, with a threshold at 2,5 ng/mL (Figure 1A). At the 10 ng/mL concentration, IL-1 already significantly increased visfatin levels at 12 h, with a maximum reached at 18 h, then followed by a slow and progressive decay at 24 and 48 h (Figure 1B). Moreover, the stimulation of HUVEC with two other vascular pro-inflammatory stimuli such as tumor necrosis factor (TNF)- (1 to 10 ng/mL 18 h) or angiotensin II (10 pmol/L to 100 nmol/L 18h) also resulted in a concentration-dependent enhancement of cellular visfatin content at thresholds of 2.5 ng/mL and 1 nmol/l, respectively (Figures 1C and 1D). Furthermore, IL-1 (10 ng/mL 18 h), selected as a model of pro-inflammatory stimulus, increased HUVEC visfatin mRNA levels, suggesting a de novo synthesis of visfatin triggered by the cytokine (Figure 2A)(PARP)-1 activation, as relevant molecules involved in inflammatory responses, was next explored. Figures 2B and 2C illustrate the stimulation of NF-B DNA binding activity and PARP-1 activity by IL-1 (10 ng/mL 18 h). respectively. Indeed, a sequential NF-B - PARP-1 activation pathway was indicated by the fact that the NF-B inhibitor PDTC (10 ol/L) abrogated PARP-1 activation by IL-1 (Figure 2C). Both NF-B and PARP-1 were necessary for visfatin induction in HUVEC, as their respective inhibitors PDTC and PJ34 (100 ol/L) prevented the increase in visfatin levels triggered by IL-1 (Figure 2D).Using immunofluorescence techniques, we observed that, under non-inflammatory conditions, visfatin predominantly exhibited a granular pattern localized within the cell nucleus (Figures 3A, left panel, and Figure 3B). However, 18 h after exposing HUVEC to IL-1 (10 ng/mL) a marked positive extranuclear filamentous staining for visfatin was also observed (Figure 3A right panel and Figure 3C). To analyse whether these filaments did co-localize with some cytoskeleton element, we used fluorescence-labelled phalloidin to detect F-actin. Figures 3A and 3C show that IL-1 promoted the colocalization of visfatin with F-actine fibers, particularly in the cytoplasm and at the cell surface (Figure 3A right pannel and 3C), suggesting a secretory pattern.To gain insight into the intracellular pathways mediating the induction of visfatin by inflammation in HUVEC, the implication of nuclear factor (NF)-B and poly (ADP ribose) polymerase Figure 2. The sequential activation of NF-B and PARP-1 activation mediates inflammation-evoked visfatin production. (A) Relative mRNA expression of visfatin in HUVEC treated with or without IL-1 (10 ng/mL 18 h). (B) NFB activation in HUVEC after incubation with IL-1 (10 ng/mL) for 1 h was determined by EMSA. Data are the meanEM of four independent experiments. P<0.05 vs basal levels. (C) Auto-modified immunoreactive PARP-1 in HUVEC stimulated for 18 with or without IL-1 (10 ng/mL 18 h), in the absence or presence of the respective PARP-1 and NF-B inhibitors PJ34 (10 ol/L) and PDTC (100 ol/L). Data are the meanEM of four independent experiments. P<0.05 vs basal P<0.05 vs IL-1. (D) Visfatin protein levels in HUVEC treated with IL-1 (10 ol/L 18 h) with or without PJ34 (10 ol/L) or PDTC (100 ol/L). Data are the meanEM of four independent experiments. P<0.05 vs basal P<0.05 vs IL-1. Representative gels are shown.Inflammation modifies the sub-cellular Figure 3. distribution pattern of visfatin in HUVEC. (A) Confocal maximum projections showing a general view of stained nuclei (blue), visfatin (green), F-actin (red), and merge (yellow) in HUVEC cultures. In non-stimulated HUVEC (left), visfatin is mainly localized within the cell nucleus, while in HUVEC stimulated with IL-1 (10 ng/mL) for 18 h (right), visfatin can be markedly found in non-nuclear localizations together with Factin. 630x magnification. (B) Magnified view of the visfatin granular nuclear distribution in non-stimulated HUVEC at 400x (left) and 1,000x (right) magnification. Nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). (C) Magnified view of the visfatin non-nuclear filamentous distribution pattern in HUVEC stimulated with IL-1 (10 ng/mL). 1,000x magnification.There has been controversy about the capacity of visfatin to be secreted to the extracellular space, as this protein lacks a signal peptide [22,23]. To explore the capacity of HUVEC to secrete visfatin, we first used an immunocytochemical approach to detect visfatin in the extracellular space [21]. As shown in Figure 4A, IL-1 promoted visfatin release, visualized as a stained halo surrounding each secreting cell. This secretion halo could not be observed in HUVEC treated with the PARP-1 inhibitor PJ34 (100 ol/L Figure 4A). As a second approach, visfatin was quantified using ELISA, and significantly higher levels were found in the supernatant of HUVEC challenged with IL-1 as compared with nonstimulated ones (Figure 4B).A few years ago, Fukuhara et al [1] first identified visfatin as a new adipocytokine identical to PBEF, a 52 kDa cytokine acting on early B-lineage precursor cells [2], and to the enzyme Nampt [3,4], which plays an essential role in the biosynthesis of NAD+ by converting nicotinamide into nicotinamide mononucleotide (NMN), which is then transformed into NAD+ by nicotinamide/nicotinic acid mononucleotide adenyltransferase (Nmnat) [24]. Despite in the last years different studies have demonstrated the capacity of exogenous visfatin to directly induce vascular cell damage [5,136,19], whether vascular cells themselves synthesize and release visfatin to the extracellular milieu has been only scarcely addressed. In the present study, immunoreactive visfatin was detected in cultured human endothelial cells. Most noticeably, the intracellular visfatin levels were markedly enhanced in response to a series of molecules implicated in vascular inflammation and disease, such as IL-1, TNF- or Ang II. Therefore, one first finding of this study was that inflamed human endothelial cells over-produce visfatin. This is most likely explained by a de novo visfatin synthesis, as indicated by the increase of visfatin mRNA levels in response to a proinflammatory stimulus. Supporting this observation, Williams et al [25] showed in a wide microarray study that visfatin was among the factors whose mRNA was increased upon IL-1 -stimulation in HUVEC, although the post-transcriptional impact of such Figure 4. Inflammation promotes visfatin secretion by HUVEC. (A) Representative microphotographs of visfatin release from HUVEC grown on Immobilon-P membranes and treated with or without IL-1, (10 ng/mL 18 h) in the absence or the presence of the PARP-1 inhibitor PJ34 (10 ol/L). Secreted visfatin appears as a diffuse halo of extracellular positive immunostaining (black arrows). Magnification 400x. (B) Visfatin content determined by ELISA in cell supernanants treated with or without IL-1 (10 ng/mL 18 h). Data are the meanEM for four independent experiments. P<0.05 vs basal finding on visfatin protein levels was not explored. Other reports using different cell types have reported visfatin production in response to inflammatory conditions such as labour, acute lung injury, sepsis or rheumatoid arthritis [269]. Moreover, here we have gained insight into the signalling mechanisms mediating visfatin induction by inflamed endothelial cells by identifying the sequential activation of NFB and PARP-1 as a key event in such process. A second main finding of the present study was that inflammation not only increased visfatin synthesis but also modified the sub-cellular distribution of visfatin while enhancing the secretion of the adipokine to the extracellular space. In line with this observation, changes in the sub-cellular visfatin distribution have been previously reported in non-proliferating PC-12 cells or confluent Swiss 3T3 fibroblasts when these cell types are activated by proliferative stimuli [22]. Indeed, two different forms of Nampt/visfatin, both intracellular and extracellular, have been identified to date. On one hand, the intracellular form would play a central role in maintaining the activity of different NAD-dependent enzymes that are implicated in the regulation of cell metabolism [4,24]. This form has been involved as a NAD supplier for a number of NADconsuming enzymes acting intra-nuclearly, which are involved in key enzymatic reactions for cell growth and survival [30,31]. In human vascular smooth muscle cells, the intracellular form has been identified as a regulator of NAD-dependent protein deacetylase activity, promoting cell maturation and increasing lifespan [32,33], while improving the functionality and angiogenic capacity, as well as its replicative lifespan, in human endothelial cells [34,35]. In this context, the present study localizes intra-nuclear Nampt/visfatin as the predominant form in non-inflamed endothelial cells, which most likely reflects cell maintenance functions. On the other hand, the extracellular form of Nampt/visfatin is synthesized and released to the extracellular milieu, where it could exert a variety of actions in a paracrine or endocrine manner [4]. Structurally, extracellular visfatin shows a slightly higher molecular weight than the intracellular isoform and seems to undergo post-transcriptional modifications [4,24]. Here, we have demonstrated the secretory activity of human endothelial cells through both the immunolocalization and quantification of visfatin in the extracellular space, which was particularly evident under inflammatory conditions. There has been controversy on the ability of visfatin to be secreted to the extracellular milieu, since this protein lacks a signal sequence for secretion [3,22,23]. However, other well-characterised peptides lacking a signal sequence for secretion, including the pro-inflammatory cytokine IL-1 itself, are released by a wide variety of cell types [36,37]. In the last years, a role for visfatin as a possible link between metabolic disorders and atherothrombotic inflammatory diseases has been supported [38]. Enhanced circulating visfatin has been proposed as an atherosclerosis marker [11,39], while other studies suggest that it rather reflects the global inflammatory status in patients with cardiovascular and renal diseases [40]. Not only circulating visfatin, but also perivascular adipose tissue-derived visfatin has been related to coronary and aortic atherosclerosis [41]. This observation highlights that locally produced visfatin may play an important paracrine role in the development of atherosclerotic lesions. In this context, activated monocytes/macrophages that closely interact with vascular cells do release visfatin [42]. Increased visfatin expression has been described in macrophages of human unstable carotid and coronary atherosclerotic plaques [10] suggesting that locally produced visfatin should be regarded as an inflammatory mediator with a role in plaque destabilization. In the present study, we have demonstrated that human endothelial cells, which constitutively express visfatin, synthesize and release significantly higher amounts of the adipokine in response to inflammation. Therefore, not only macrophages and perivascular adipose tissue, but also the inflamed endothelium itself represents a local source of visfatin that may promote and amplify vascular damage. In conclusion, human endothelial cells synthesize and secrete visfatin, which is particularly reinforced in a proinflammatory environment. Whilst acknowledging the limitations of an in vitro study, we propose that visfatin released by endothelial cells may act as a local pro-inflammatory mediator in the vascular wall with a potential role in atherothrombotic diseases.Staphylococcus aureus generally colonizes human skin and mucous membranes. The anterior nares are most frequently the site of carriage [1,2]. Approximately 30% of the general population will be colonized by S. aureus at one time point [3] and three types of carriership can be distinguished: persistent carriers (~20%), intermittent carriers (~30%) and non-carriers (~50%) [3-6]. Nasal colonization by S. aureus is one of the most important factors in the pathogenesis of nosocomial infections, mainly in surgical site and vascular device related infections [7-10]. Staphylococcal infections normally occur when the mucosal or skin barrier breaches, e.g., by scratching, mechanical stress or surgery, thereby allowing access to the adjoining tissues or the bloodstream [11]. The change of the bacteria from colonizers to pathogens is accompanied by a drastic change in expression profiles [12]. Similar changes can be expected when bacteria change hosts. These changes in expression profile may be due to environmental signals, but also due to mutational changes. For Group A Streptococci (the causative agent of scarlet fever, necrotizing fasciatis and other infections) it was shown that an insertion of 7 nucleotides in a regulator gene was sufficient to alter transcription and turn a benign Group A Streptococcus strain into a highly infectious variant [13].Whole genome sequencing of sets of S. aureus isolates obtained from colonization and subsequent infection from the same patient may provide insight into small changes (single nucleotide polymorphisms or SNPs) in the genome that may contribute to the altered gene expression profiles during infection when compared to colonization [14]. The transfer of livestock-associated methicillin-resistant S. aureus (LA-MRSA) from pigs to humans is the most common change of hosts among S. aureus as up to 30% of the pig farmers are at one moment colonized by LA-MRSA [15]. We therefore compared the whole genome sequences of three sets of isolates from the same patients as well as a set of isolates from a colonized pig and a farmer obtained from the same farm. 2909748The data indicate that differences between the isolate from infection and the colonizing isolate exist, although one set of human isolates did not constitute a true pair. Some differences between the LAMRSA isolates was also observed.Isolates were obtained as part of routine diagnostic testing and were analyzed anonymously and the isolates, not humans, were studied. All data was collected in accordance with the European Parliament and Council decision for the epidemiological surveillance and control of communicable disease in the European community [16,17].
