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      Maslinic acid alleviates ischemia/reperfusion-induced inflammation by downregulation of NFκB-mediated adhesion molecule expression

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          Ischemia/reperfusion (I/R)-induced inflammation is associated with enhanced leukocyte rolling, adhesion and transmigration within the microcirculation. These steps are mediated by hypoxia-triggered signaling pathways, which upregulate adhesion molecule expression on endothelial cells and pericytes. We analyzed whether these cellular events are affected by maslinic acid (MA). Mitochondrial activity and viability of MA-exposed endothelial cells and pericytes were assessed by water-soluble tetrazolium (WST)-1 and lactate dehydrogenase (LDH) assays as well as Annexin V/propidium iodide (PI) stainings. Effects of MA on hypoxia and reoxygenation-induced expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by flow cytometry. The subcellular localization of the NFκB subunit p65 was analyzed by immunofluorescence and Western blot. I/R-induced leukocytic inflammation was studied in MA- and vehicle-treated mouse dorsal skinfold chambers by intravital fluorescence microscopy and immunohistochemistry. MA did not affect viability, but suppressed the mitochondrial activity of endothelial cells. Furthermore, MA reduced adhesion molecule expression on endothelial cells and pericytes due to an inhibitory action on NFκB signaling. Numbers of adherent and transmigrated leukocytes were lower in post-ischemic tissue of MA-treated mice when compared to vehicle-treated controls. In addition, MA affected reactive oxygen species (ROS) formation, resulting in a diminished oxidative DNA damage. Hence, MA represents an attractive compound for the establishment of novel therapeutic approaches against I/R-induced inflammation.

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          Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers.

          Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.
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            Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo

            Transmigration of neutrophils from the vascular lumen through venular walls into the surrounding tissue is a vital response in host defense reactions during injury and infection and also a major cause of numerous inflammatory disorders. The venular wall is composed of two cellular components, endothelial cells (ECs) and pericytes, and a noncellular matrix protein structure called the vascular basement membrane (BM), which is generated by both ECs and pericytes (Nourshargh et al., 2010). In recent years, our understanding of the mode and molecular pathways mediating neutrophil migration through ECs has substantially enhanced (Ley et al., 2007; Muller, 2009). Although this response can occur relatively rapidly in vivo (∼4–6 min; Woodfin et al., 2011), full breaching of the venular wall takes significantly longer (∼15–40 min; Katori et al., 1990; Ley et al., 1993; Yadav et al., 2003), suggesting a considerable transit time through other components of the vessel wall. In this context, despite recent insights into leukocyte–BM interactions (Rowe and Weiss, 2008; Nourshargh et al., 2010), at present almost nothing is known about neutrophil–pericyte interactions in vivo and the potential role of pericytes in leukocyte transmigration. These key issues were the focus of the present investigation. Pericytes are long cells (∼70 µm in length) surrounding the EC layer of capillaries, postcapillary venules, and collecting venules, and are embedded within the venular BM (Shepro and Morel, 1993; Hirschi and D’Amore, 1996). Pericytes are closely associated with the endothelium, and play a critical role in maintaining the integrity of the vessel wall and contributing to the generation of the venular BM (Cohen et al., 1980; Mandarino et al., 1993; Armulik et al., 2005; Edelman et al., 2006). Pericytes exhibit morphological and phenotypic differences depending on vessel type, vascular bed, developmental stage, species, and pathological conditions (Hirschi and D’Amore, 1996; Sims, 2000), rendering their study in vivo and culture in vitro complex. As a result, the role of pericytes in inflammation and leukocyte trafficking remains largely unexplored. In most tissues, the pericyte network is discontinuous around the endothelium of postcapillary venules, with gaps between adjacent cells. The extent of pericyte coverage around microvessels varies substantially in different organs, suggesting different properties in different tissues. Previous studies from our group have shown that neutrophils breach the pericyte layer by migrating through gaps between adjacent cells, sites that are aligned with regions within the venular basement membrane where there is lower deposition of certain BM constituents (Wang et al., 2006; Voisin et al., 2009, 2010). To further investigate the mechanisms and dynamics of neutrophil migration through the pericyte sheath, in this study we have used numerous imaging modalities (including three-dimensional [3D] real-time imaging) to analyze pericyte morphology and neutrophil–pericyte interactions in inflamed cremasteric venules. The results show that pericytes actively support neutrophil migration through venular walls and that blockade of neutrophil crawling on pericytes inhibits neutrophil transmigration. Specifically, we observed that neutrophils exhibited sub-EC crawling along pericyte processes to gaps between adjacent pericytes. The directionality and speed of this crawling response within venular walls, termed abluminal crawling, appeared to be supported by pericyte-expressed intercellular adhesion molecule-1 (ICAM-1) and its leukocyte integrin ligands, Mac-1 and LFA-1. Finally, we provide evidence for the ability of pericytes to exhibit shape change resulting in enhanced mean gap size between adjacent cells. These sites were preferentially used as exit points by transmigrating neutrophils in response to the proinflammatory cytokines TNF and IL-1β. This effect appeared to be mediated via direct stimulation of pericytes, as these cells were found to express receptors for both TNF and IL-1β. Collectively, the study identifies several modes through which pericytes support neutrophil migration through venular walls, adding additional steps to the leukocyte adhesion cascade that describes the process of leukocyte infiltration into inflamed tissues. RESULTS Four-dimensional (4D) imaging of neutrophil–pericyte interactions in vivo To facilitate our studies, transgenic αSMA-RFPcherry mice were bred with Lys-EGFP-ki mice (Faust et al., 2000), resulting in the generation of a novel mouse colony that expressed RFPcherry pericytes and enhanced GFP (EGFP) neutrophils and monocytes. The application of confocal intravital microscopy (IVM) to analyze inflammatory events within cremasteric venules of these mice resulted in a uniquely powerful model for the study of neutrophil–pericyte interactions in real-time in three dimensions (Fig. 1, A and B; and Video 1). The image acquisition settings were optimized for tracking of GFPhigh neutrophils, with GFPlow monocytes being barely visible (Woodfin et al., 2011). Furthermore, in vivo immunostaining of EC junctions using an anti–platelet EC adhesion molecule-1 (PECAM-1) mAb (clone 390, which was previously shown not to effect leukocyte transmigration; Christofidou-Solomidou et al., 1997; Woodfin et al., 2011) allowed for tracking of neutrophils through both the endothelium and pericytes (Fig. 1 C). The model enabled us to observe neutrophil behavior at different stages of their migration through venular walls and into the interstitial space (Fig. 1 C). In investigating TNF-stimulated tissues, the following steps were observed and analyzed: (a) migration of neutrophils through the endothelium (largely through EC junctions; trans-EC migration [TEM]); (b) crawling of neutrophils within the venular wall (abluminal crawling); (c) migration of neutrophils through gaps between adjacent pericytes; and (d) migration of leukocytes into the interstitial tissue (Fig. 1 D). Neutrophils were found to have distinct morphological profiles at these different steps, e.g., significant flattening, reduced sphericity, and reduced elongation when crawling within the venular wall (abluminal crawling), as compared with leukocytes exhibiting luminal crawling (unpublished data). These parameters enabled us to clearly identify and analyze the dynamics of neutrophil abluminal behavior after TEM and when in contact with the pericyte sheath. Figure 1. 4D imaging of neutrophil transmigration. αSMA-RFPcherry ×Lys-EGFP-ki mice exhibiting endogenously labeled pericytes (RFPcherry, red) and neutrophils (EGFP, green) were subjected to EC junctional labeling using a directly conjugated Alexa Fluor 647 nonblocking mAb to PECAM-1 mAb (blue). (A) Composition of six 3D-reconstructed confocal images of the cremasteric microcirculation 120 and 240 min after intrascrotal injection of TNF. a, arteriole; c, capillary; cp, capillary pericyte; pcv, postcapillary venule. Bar, 100 µm. (B) Still images of a cremaster muscle postcapillary venule showing the development of an inflammatory reaction at the indicated time points after stimulation with TNF. Bar, 10 µm. (C) The images in the top panels are 3D reconstructions of half postcapillary venules showing a luminal neutrophil at an early stage of breaching the endothelium (cell 1) and a neutrophil that has already crossed the endothelium and is between the endothelium and pericyte layer (cell 2) at 3 h after TNF stimulation. The middle and bottom panels show the position of the indicated neutrophils from the luminal side (left hand side) or from 2-µm cross-sections of venules (right hand side) along the indicated lines demonstrating the position of the leukocyte relative to ECs and the pericyte layer. Bar, 10 µm. (D) Using the 3D real-time imaging model to analyze neutrophil transmigration through venular walls, several distinct steps were observed as illustrated in the diagram: (1) TEM, (2) motility between endothelium and the pericyte sheath (abluminal crawling), (3) migration through gaps between adjacent pericytes, and (4) interstitial migration. Images are representative of at least six separate experiments. Analysis of the dynamics of different transmigration steps showed that in response to TNF, neutrophils breached ECs rapidly (average duration of ∼4 min), which is in line with TEM dynamics observed when using other stimuli (Woodfin et al., 2011). After TEM, neutrophils exhibited significant venular wall (abluminal) crawling, covering an average distance of ∼54 µm within a period of 20 min before breaching the pericyte sheath by migrating through gaps between adjacent cells with a duration of 11.1 ± 0.8 min. Crossing the venular walls from the first captured frame showing a leukocyte entering an EC junction to an EC entering the interstitial space took ∼35 min. Overall, these results show the development of a uniquely powerful imaging method for direct observation of neutrophil–pericyte interactions in real time, enabling investigations of the profile, dynamics, and mechanisms of neutrophil migration through venular walls beyond TEM. ICAM-1, Mac-1, and LFA-1 mediate neutrophil crawling on pericytes The aforementioned imaging method was applied to a more in-depth investigation of neutrophil–pericyte interactions in 4D. Tracking neutrophils beyond TEM demonstrated that the motility of neutrophils within venular walls was governed by the morphology of pericytes in that leukocytes used pericyte processes as tracks for their crawling behavior (Fig. 2, Video 2, Video 3, Video 4, and Video 5). Although neutrophils crawled an average distance of 54.4 ± 3.9 µm on pericytes before breaching the pericyte layer, the displacement length covered (i.e., the minimum distance between the TEM site and the eventual pericyte gap used by neutrophils to breach the pericyte layer) was on average 17.3 ± 0.9 µm. The significant difference in the quantified total migration distance and straight displacement distance is accounted for by the meandering nature of this motility response within the curved venular wall (Fig. 2 C). In some instances, multiple neutrophils were observed to be following each other within the venular wall to the same pericyte gap or along the same pericyte processes (see following paragraph). Interestingly, the majority of neutrophils tracked for abluminal crawling were seen to use pericyte processes as substrate (approximately >98%) and avoided gaps between adjacent pericytes (Fig. 2). Figure 2. Analysis of neutrophil abluminal crawling in vivo. Cremaster muscles of αSMA-RFPcherry x Lys-EGFP-ki mice were stimulated with TNF for 2 h before being exteriorized and visualized by live confocal microscopy. (A) Time-lapse images showing a neutrophil (green) crawling along pericyte processes (red) from its site of TEM (i.e., starting point of the time course analysis, blue arrow) toward a gap between adjacent pericytes (red arrow). In the top panels, the neutrophil and pericyte layer are viewed from outside the vessel. The crawling path is shown with the yellow line. An isosurface mask was created to enable clearer visualization of the migrating neutrophil within the pericyte layer. In the bottom panels, 2-µm cross-sections in the z-axis show the position of the migrating neutrophil relative to the EC layer (blue) in the first image showing that it has passed the endothelium. The subsequent images show the neutrophil in relation to pericytes (red) during its abluminal crawling as captured at multiple time points. (B) Time-lapse images showing another example of neutrophil crawling along pericyte processes and avoiding the gaps between adjacent pericytes. Crawling path (yellow line) and directionality (black arrow) are shown in the top panels; the vessel segment is viewed in 3D from outside the vessel. The bottom panels show a higher magnification maximal intensity projection (2D) of the vessels with the shape of the neutrophil drawn with a green dotted line at each of the time points to illustrate its shape and its position relative to pericyte processes. (C) Four examples of postcapillary venules (pericyte in red) viewed in 3D from the extravascular space (top) or at a z-section angle (bottom), together with individual crawling paths (green line) and directionality (black arrow) of the neutrophil being tracked along pericyte processes in 3D. For clarity, the neutrophil being tracked is not depicted. Examples 1 and 2 show both the migration path and directionality of cells being tracked in A and B, respectively. All corresponding track and displacement lengths are given below the images. Bars, 10 µm. As neutrophil intravascular crawling is reportedly ICAM-1 and Mac-1 dependent (Phillipson et al., 2006), we hypothesized that abluminal neutrophil crawling on pericytes may also be mediated by similar mechanisms. In initial studies, we investigated the expression of ICAM-1 on venular pericytes in vivo. Immunostaining of fixed cremaster muscles for ICAM-1 indicated a significant up-regulation in the expression of this adhesion molecule on both ECs and pericytes of TNF-stimulated venules as compared with unstimulated tissues (Fig. 3, A and B). Furthermore, local administration of functional blocking anti–ICAM-1 mAb and anti–Mac-1 mAbs (injected 2 h after TNF stimulation) impaired neutrophil crawling on pericytes as compared with responses detected in control mAb-treated tissues (Fig. 3, C and D). Specifically, in anti–ICAM-1 mAb-treated cremaster muscles, abluminal neutrophils exhibited reduced speed and length of crawling within venular walls, resulting in cells occasionally “moving on the spot,” (i.e., exhibiting multiple oscillatory movements without significant displacement [ 50 µm2). Figure 5. Neutrophils migrate through enlarged pericyte gaps. (A) The graph shows the frequency (percentage) of different sized gaps between adjacent pericytes in cremasteric postcapillary venules (unstimulated and TNF-stimulated) as detected by ex vivo immunofluorescence labeling and confocal microscopy. Gap sizes in the depicted range (1–52 µm2) represent ∼97% of all detected gaps (at least 4 venules per animal analyzed in 5 mice). The boxed region indicates the range of gaps used by ∼70% of observed transmigration events as analyzed by 4D confocal IVM (significantly different from the frequency of transmigration events detected in the nonboxed range; ***, P 70% of these “KC/ICAM-1 high” borders were associated with pericyte gaps of 8–50 µm2 size, a gap size range preferentially used by transmigrating neutrophils. These associations suggest that expressions of KC and/or ICAM-1 near pericyte gaps may guide neutrophils to exit sites within the pericyte sheath. Although this presents a tantalizing possibility, at present we are unable to confirm this hypothesis in real-time neutrophil-tracking studies because of the lack of availability of a nonblocking anti–ICAM-1 mAb that can be used for in vivo labeling of pericyte ICAM-1. Pericytes are contractile cells and, as such, several studies have shown the ability of pericytes to exhibit shape change after stimulation with vasoactive mediators in vitro, such as histamine (Murphy and Wagner, 1994; Speyer et al., 1999). As pericyte shape change appeared to facilitate neutrophil migration through the pericyte sheath, this response was investigated in more detail. Cremaster muscles or ear skin stimulated with TNF and IL-1β showed significantly enlarged gaps between adjacent pericytes, increasing by ∼80% after a 2–4-h stimulation. Full time course analysis of pericyte shape change and neutrophil transmigration indicated that in response to TNF, the former preceded the response of neutrophil transmigration, suggesting that pericyte shape change occurs independently of neutrophils. This is in contrast to inflammation-induced remodeling of BM permissive regions (LERs), a response that we have previously shown to be strictly neutrophil dependent (Voisin et al., 2009, 2010; Wang et al., 2006). The ability of pericytes to exhibit shape change in a neutrophil-independent manner was conclusively demonstrated in studies where TNF and IL-1β were found to induce enlarged pericyte gaps in both control and neutrophil-depleted animals. To investigate the mechanism of this response, the potential expression of receptors for TNF and IL-1β on pericytes was explored. Immunofluorescence staining of unstimulated cremaster muscles revealed significant levels of TNFRI, TNFRII, and ILRI on pericytes comparable to levels detected on ECs. Parallel experiments conducted with the pericyte-like cell line C3H/10T1/2 (Reznikoff et al., 1973) showed the ability of these cells to exhibit change in morphology in response to TNF and IL-1β and to express TNFRI, TNFRII, and ILRI. Collectively, the present results demonstrate that pericytes express receptors for TNF and IL-1β and that activation of these receptors can lead to increased expression of ICAM-1 and KC that support neutrophil abluminal crawling after TEM to preferred pericyte gaps. However, our results show that pericyte shape change as induced by TNF and IL-1β in vivo can contribute to the generation of enlarged gaps between adjacent pericytes that are preferentially used by transmigrating neutrophils. Details of the signaling pathways that regulate pericyte shape change in vivo are at present unclear, but both TNF and IL-1β are known to activate small GTPases that play a key role in actin cytoskeleton rearrangement (Puls et al., 1999), and both cytokines have previously been reported to induce morphological changes in rat lung pericytes in vitro (Kerkar et al., 2006). In addition, the disassembly of αSMA stress fibers in isolated bovine retina pericytes causing reduced cell size in vitro is reportedly mediated via the small GTPase RhoA (Kolyada et al., 2003; Kutcher et al., 2007; Kutcher and Herman, 2009), but the potential role of such pathways in vivo has yet to be clarified. In summary, through development of an imaging platform that allows direct quantitative analysis of neutrophil–pericyte interactions, we have unraveled several modes through which pericytes can facilitate neutrophil transmigration through venular walls. Hence, in addition to their accepted role in regulation of vascular tone, integrity, and barrier function (Shepro and Morel, 1993; Hirschi and D’Amore, 1996; Edelman et al., 2006), the present results demonstrate a role for pericytes in inflammation, indicating the need for further analysis of the role of these mural cells in vascular functions. MATERIALS AND METHODS Reagents. Recombinant murine TNF and IL-1β were purchased from R&D Systems. The neutrophil-depleting monoclonal rat anti–mouse Ly-6G and Ly-6C (GR1) antibody (clone RB6-8C5, NA/LE) was purchased from BD. The following primary antibodies were used for immunofluorescence labeling for confocal imaging and confocal IVM: monoclonal mouse anti–mouse αSMA-Cy3 (clone 1A4; Sigma-Aldrich); monoclonal rat anti–mouse ICAM-1 (clone YN1/1.4.7; eBioscience); monoclonal rat anti–mouse PDGFRβ (clone APB5), anti–mouse Mac-1 (clone M1/70), and anti–mouse LFA-1 (clone M17/4; BioLegend); monoclonal Armenian hamster anti–mouse IL-1RI (clone JAMA-147 used for flow cytometry; BioLegend); polyclonal goat anti–mouse IL-1RI (used for confocal microscopy; R&D Systems); monoclonal rat anti–mouse MRP-14 (clone 2B10; a gift from N. Hogg, Cancer Research UK, London, UK; Hobbs et al., 2003); nonblocking monoclonal rat anti–mouse PECAM-1 (clone C390; generated as described in Christofidou-Solomidou et al., 1997) directly conjugated to Alexa Fluor 647 using Molecular Probes Alexa Fluor Monoclonal Antibody Labeling kit (Invitrogen); monoclonal Armenian hamster anti–mouse TNFRI (clone 55R-286 used for flow cytometry; BioLegend); polyclonal goat anti–mouse TNFRI (used for confocal microscopy; R&D Systems); monoclonal Armenian hamster anti–mouse TNFRII (clone TR75-89 used for flow cytometry; BioLegend); polyclonal goat anti–mouse TNFRII (used for confocal microscopy; R&D Systems); rat anti–mouse KC (clone 124014 used for confocal microscopy; R&D Systems). The following purified antibodies were used as isotype-matched controls: Armenian hamster IgG1 (BD), goat IgGs (R&D Systems), mouse IgGs (BD), or rat IgG2a or IgG2b (AbD Serotec). Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen) or biotin (BioLegend) were used if necessary. The biotinylated anti–Armenian hamster secondary antibody was detected using streptavidin conjugated with Alexa Fluor 488 (Invitrogen). Animals. Male (20–25 g) mice from several colonies were used in this study. Pure WT C57BL/6 mice were purchased from Harlan-Olac. The αSMA-RFPcherry mice are transgenic animals in which the RFP variant cherry transgene was inserted and expressed upon the αSMA promoter, resulting in red fluorescent pericytes and smooth muscle cells. This mouse strain is on a C57BL/6 background and was generated according to established protocols previously detailed for generation of αSMA-EGFP transgenic mice (Yokota et al., 2006). The expression of RFPcherry was largely as found in the αSMA-EGFP transgenic animals. In brief, the regulatory sequence of αSMA gene promoter used contains −1,074 bp of the 5′ flanking region, the transcription start site, 48 bp of exon 1, the 2.5-kb intron 1, and the 15-bp exon 2 of mouse αSMA (Wang et al., 1997). This sequence was fused with the sequence of the red fluorescent protein RFPcherry (Shaner et al., 2004). Transgenic DNA purification and microinjection of the DNA were performed as described previously (Schlaeger et al., 1995). The Lys-EGFP-ki mice (backcrossed on C57BL/6 background for at least 8 generations; Faust et al., 2000), in which the gene for EGFP has been knocked into the lysozyme M (lys) locus, exhibit fluorescent myelomonocytic cells, with mature neutrophils comprising the highest percentage of EGFP high cells. These mice were used with the permission of T. Graf (Albert Einstein College of Medicine, Bronx, NY) and were provided for the study by M. Sperandio (Ludwig Maximilians University, Munich, Germany). Finally, the novel αSMA-RFPcherry×Lys-EGFP-ki mouse strain was generated in-house by interbreeding the αSMA-RFPcherry transgenic mice with Lys-EGFP-ki animals, yielding a valuable mouse colony expressing red pericytes and green leukocytes (neutrophils and monocytes) that were used for real-time confocal microscopy technique. Only mice heterozygote for Lys-EGFP-ki that showed normal transmigration responses in vivo (Woodfin et al., 2011) were used in the study, and thus no other littermates or WT mice were needed as controls. All animals were housed in individually ventilated cages and facilities were regularly monitored for health status and infections. All experiments were performed under the UK legislation for the protection of animals, and at the end of all in vivo procedures involving anesthesia, animals were humanely killed by cervical dislocation in accordance with UK Home Office regulations. Induction of inflammatory reactions in the murine cremaster muscle. The cremaster muscle was used as the principal tissue for analysis of leukocyte–vessel wall interactions because of its thin and transparent nature, which allows it to be whole mounted for immunofluorescence staining and confocal microscopy. For this purpose, mice were sedated via intramuscular (i.m.) injection of 1 ml/kg anesthetics (40 mg ketamine and 2 mg xylazine in saline), and subsequently stimulated locally via intrascrotal (i.s.) injection of TNF (300 ng/400 µl saline) or IL-1β (50 ng/400 µl saline). As vehicle control, mice received i.s. injection of 400 µl saline. Different in vivo test periods were investigated as governed by the time course of action of these stimuli and the experimental objective. At the end of the in vivo test periods, mice were either sacrificed and cremaster muscles were dissected away and fixed for subsequent ex vivo analysis using immunofluorescence labeling and confocal microscopy, or mice were anaesthetized via i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) in saline. Cremasters were surgically exteriorized for in vivo analysis using confocal IVM, as previously described (Woodfin et al., 2011). Induction of inflammatory reactions in the mouse ear skin. Pure C57BL/6 animals were sedated and injected intradermally (i.d.) with TNF (150 ng/30 µl PBS), IL-1β (10 ng/30 µl PBS), or 30 µl PBS (vehicle control) in the ear. At the end of the experiments, mice were sacrificed and ears were dissected away for subsequent ex vivo analysis by immunofluorescence labeling and confocal microscopy, as previously described (Voisin et al., 2010). Neutrophil depletion. Pure C57BL/6 mice were depleted of their circulating neutrophils by i.p. injection of anti-GR1 antibody (100 µg/1 ml saline) 24 h before i.s. administration of TNF or IL-1β. Control mice received the rat IgG2b isotype-matched control antibody. To determine the level of neutrophil depletion, blood neutrophil counts were performed as previously described (Wang et al., 2006). In brief, tail vain blood samples were collected before and 24 h after antibody administration, indicating an average neutrophil depletion of >80%. This almost total depletion of neutrophils in GR1-treated mice was also confirmed by confocal microscopy analysis of TNF-stimulated cremaster muscles stained for MRP-14 (neutrophil marker). Immunofluorescence labeling and confocal microscopy analysis of whole-mounted murine tissues. Pure C57BL/6 mice were sacrificed and cremaster muscles or ears were dissected away and fixed in 100% methanol or 4% paraformaldehyde (PFA) in PBS for 30 min at 4°C. Subsequently immunofluorescence staining and confocal analysis of whole mounted tissues was performed as previously described (Voisin et al., 2010). In brief, to visualize pericytes (anti-αSMA antibody) and/or neutrophils (anti–MRP-14 antibody) and the cytokine receptors IL-1RI, TNFRI, TNFRII, and the chemokine KC, tissues were blocked and permeabilized in PBS containing 10% normal goat serum or rabbit serum (depending on the species of secondary antibodies used), 10% FCS, and 0.5% Triton X-100 for 2 h at room temperature. This was followed by incubation with the primary antibodies or appropriate control antibodies in PBS plus 10% FCS overnight at 4°C. If necessary, tissues were incubated with specific Alexa Fluor–conjugated secondary antibodies in PBS plus 10% FCS for 3 h at 4°C. The samples were imaged at 20°C using a LSM 5 PASCAL laser-scanning confocal microscope (Carl Zeiss) incorporating a 40× water-dipping objective (numerical aperture (NA) 0.75) or a 63× oil-immersion objective (NA 1.4). Z-stack images of postcapillary venules (within a diameter of 20–50 µm) were captured using the multiple track scanning mode at every 1 µm (40×) or 0.4 µm (63×) of tissue depth at a resolution of 1,024 × 1,024 pixels in the x × y plane, corresponding to a voxel size of ∼0.26 × 0.26 × 1/0.4 µm in x × y × z, respectively. Confocal gain, offsets, and laser power were first set on samples stained with isotype control antibodies before being applied for analysis of tissues stained for specific molecules of interest. Resulting confocal images were then analyzed using 3D reconstruction software such as ImageJ (National Institutes of Health) and IMARIS (Bitplane). Postcapillary venules, expressing pericytes and not smooth muscle cells, were identified by vascular morphology, localization within the microcirculation and flow profile. The identification of pericytes in these vessels by this mode was confirmed by immunostaining of tissues for pericyte markers such as PDGFRβ, SMA22α, and NG2. Neutrophil transmigration was quantified using IMARIS, as previously described (Wang et al., 2006; Voisin et al., 2010). To quantify the size of gaps between adjacent pericytes confocal images were 3D reconstructed and split in half in silico along the longitudinal vessel axis using ImageJ. Resulting image sections of semivessels were transformed into grayscale intensity projection and gaps between adjacent pericytes, as detected by αSMA-negative regions, were measured manually. Unstimulated and saline-injected samples were pooled because of identical results. To assess ICAM-1, PDGFRβ, KC, IL-1RI, TNFRI, and TNFRII expression on postcapillary venules, the mean fluorescence intensity (MFI) of tissues stained with control isotype-matched antibodies and tissues stained for specific molecules of interest were determined using IMARIS software. To analyze ECs and pericytes separately, an isosurface was created representing exclusively the endothelium or the pericyte sheath. This parameter depicts the limitation of the volume of interest in which IMARIS determines the MFI of other channels. Isosurfaces embodying the EC or pericyte layer were built by using the channel showing immunofluorescence labeling of PECAM-1 or αSMA as a source, respectively. PECAM-1, ICAM-1, or PDGFRβ labeling was achieved by i.s. injection of 2, 5, or 10 µg anti–PECAM-1-Alexa Fluor 647, anti–ICAM-1-Alexa Fluor 488, or purified anti-PDGFRβ antibodies, respectively, in 400 µl saline. 30 min to 2 h later, mice were sacrificed, the vasculature was washed using 10 ml saline (containing 10% heparin), and cremaster muscles were dissected from the mice for subsequent ex vivo whole-mount immunofluorescence staining. Quantification of expression of molecules of interest was expressed as the MFI for specifically stained tissues minus the MFI of samples stained with an appropriate isotype control antibody. Analysis of expression levels of ICAM-1 and KC in pericyte gap borders. To quantify the expression levels of ICAM-1 and KC on pericyte cell body and pericyte gap borders, 3D images of cremaster tissues labeled for αSMA, PECAM-1, and ICAM-1 or KC, as described in the previous section, were analyzed by IMARIS and Image J software. In brief, an isosurface was produced from the PECAM-1 channel and a new channel-mask of ICAM-1 positive, PECAM-1 negative voxels (i.e., ICAM-1 staining outside PECAM-1 volume) was created from this isosurface. A second isosurface from αSMA channel was then generated and another new channel-mask for ICAM-1 positive, αSMA positive, PECAM-1 negative voxels (i.e., ICAM-1 staining in αSMA volume excluding PECAM-1 volume) from this isosurface was created. 2D projection images were then generated on the αSMA channel and the new ICAM-1–positive, αSMA-positive, PECAM-1–negative channel mask. Images of half vessels were then imported into Image J software to measure the area and intensity of ICAM-1 in both gap borders and pericyte sheath. Borders of pericyte gaps were defined as regions within the αSMA channel 2 µm away from the gap border. Area of pericyte gaps was manually measured as described in the previous section. Confocal IVM. Confocal IVM was used to directly observe changes in pericyte morphology and to study leukocyte/pericyte interactions within cremasteric postcapillary venules upon TNF stimulation in real-time in 3D, using αSMA-RFPcherry x Lys-EGFP-ki mice. To elicit leukocyte recruitment and enlargement of gaps between adjacent pericytes, animals were sedated via i.m. injection of 1ml/kg anesthetic (40 mg/kg ketamine and 2 mg/kg xylazine in saline) and were subsequently subjected to i.s. injection of TNF (300 ng/400 µl saline). TNF was co-administered with a nonblocking anti–mouse Alexa Fluor 647–labeled PECAM-1 antibody (clone C390, 2 µg) to visualize the endothelium as previously described (Woodfin et al., 2011). Specificity of this EC junctional staining was confirmed using PECAM−/− mice and an isotype control antibody. 2 h later, mice were prepared for confocal IVM as previously detailed (Woodfin et al., 2011). In brief, mice were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and maintained at 37°C on a custom-built, heated microscope stage. The cremaster muscle was exteriorized, pinned out flat over the optical window of the stage, and superfused with warm Tyrode’s solution. During confocal IVM, z-stack images of a postcapillary venule (analyzed vessels were largely within a diameter of 20–50 µm) were captured every minute using a Leica SP5 confocal microscope (Leica) incorporating a 20× water-dipping objective (NA 1.0) for 2 h. Images were acquired with sequential scanning of different channels at a resolution of 1,024 × 512 pixels in the x × y plane and 0.7-µm steps in z-direction, corresponding to a voxel size of ∼0.23 × 0.23 × 0.7 µm in x × y × z, respectively. Throughout the experiment, cremaster muscles were constantly superfused with warm Tyrode’s salt solution. For ICAM-1–, Mac-1–, and LFA-1–blocking experiments, blocking antibodies or rat IgG2b (anti–ICAM-1/Mac-1 isotype) or IgG2a (anti–LFA-1 isotype) control antibodies were administered (i.s. 50 µg) 2 h after TNF stimulation and 30 min before exteriorization of the cremaster muscle for confocal IVM. This protocol did not effect the early luminal neutrophil-EC interactions, and thus enabled the analysis of neutrophil responses after TEM. To validate the inhibitory effect of the blocking anti–ICAM-1 mAb, in some experiments the anti–ICAM-1 mAb was co-injected locally with TNF. In these studies, the anti–ICAM-1 mAb significantly inhibited neutrophil adhesion to ECs within the vascular lumen and by extension markedly suppressed neutrophil transmigration. To control for potential Fc binding effects of the anti–ICAM-1 mAb on neutrophil crawling parameters, the effect of a rat anti–mouse PDGFRβ mAb (pericyte binding) was also tested using the same dose and injection protocol. Two nonbinding rat IgG isotype control antibodies (IgG2a and IgG2b) were also tested and, collectively, no differences were detected between untreated and control mAb-treated (including the anti-PDGFRβ mAb) groups. The resulting 4D confocal image sequences were analyzed offline using IMARIS software, which renders the optical sections of half vessels into 3D models, thereby enabling the dynamic interaction of leukocytes and the vessel wall to be observed, tracked, and analyzed. All images and videos show half vessels to enable clear visualization from only one luminal or abluminal side of the vessel wall. Analysis of neutrophil behaviors. Neutrophil responses in 4D were quantified using the IMARIS software through analysis of 4D confocal image sequences obtained via confocal intravital. Specifically, parameters were established for investigating the profile and dynamics of neutrophil transmigration through the endothelial layer and the pericyte sheath. For each image sequence, only transmigration events were analyzed that were clearly visible in terms of their location and dynamics and were imaged in full in terms of TEM and transmigration through the pericyte sheath. Here, recordings were conducted on half-vessels to enable clear visualization of migratory events through the different components of the venular wall in 3D. On average, 70 migratory events were analyzed from 4–7 mice per group. The duration of TEM was quantified from the first frame showing a disruption of PECAM-1 labeling to the frame in which the neutrophil had fully transmigrated through the endothelium, as previously described (Woodfin et al., 2011). From this frame onwards, abluminal crawling was analyzed to the frame in which the neutrophil had found the gap in the pericyte layer that was used for breaching the pericyte sheath. Duration of transmigration through the pericyte layer was quantified starting from this frame to the frame in which the neutrophil had fully transmigrated through the gap and entered the extravascular tissue. Individual neutrophil migration paths were tracked manually using IMARIS and the generated cell tracks were subsequently used to quantify the following parameters: (a) duration of transmigration (minutes), (b) speed of migration (micrometers/second), (c) track length (micrometers), (d) venular wall track displacement length (i.e., length in micrometers of the shortest connection between the end of TEM to the point when a neutrophil initiates to breach the pericyte layer), and (e) track straightness (ratio of track displacement length to track length). Other parameters of cell morphology, such as leukocyte sphericity, flattening, and elongation, were also quantified using IMARIS software after the generation of an isosurface on neutrophils at different stage of their migration (i.e., cells adherent or crawling luminally along the endothelium, abluminal crawling between ECs and pericytes, and when fully migrated into the extravascular space). This model was also used to quantify the size of pericyte gaps used by transmigrating neutrophils to breach the pericyte layer. Culture of C3H/10T1/2 cells. The cell line C3H/10T1/2 (clone 8) was purchased from the European Collection of Cell Cultures. These murine fibroblasts isolated from a line of C3H mouse embryo cells (Reznikoff et al., 1973) show pericyte/smooth muscle cell–like properties and express markers of the pericyte/SMC lineage such as αSMA, PDGFRβ, SM22α, and NG2 (as shown by flow cytometry). C3H/10T1/2 cells were cultured at subconfluent density according to the supplier’s protocol. Flow cytometry. C3H/10T1/2 cells were harvested using 0.02% EDTA and were subsequently immunostained for IL-1RI, TNFRI, TNFRII, or an Armenian hamster isotype control antibody. The cells were then incubated with specific anti–hamster secondary antibodies conjugated with biotin. The biotinylated anti–Armenian hamster secondary antibody was detected using streptavidin conjugated with Alexa Fluor 488. Samples were analyzed using a FACSCalibur flow cytometer (BS) and the flow cytometry analysis software FlowJo. Transfection of C3H/10T1/2 cells with Lifeact-eGFP. To analyze shape change of C3H/10T1/2 cells in response to inflammatory cytokines, fluorescence time-lapse microscopy was performed in cells transfected with the F-actin probe Lifeact-eGFP (Riedl et al., 2008; a gift from M. Sixt, Institute of Science and Technology, Klosterneuburg, Austria) using established protocols (Alexopoulou et al., 2008). In brief, pmeGFP-N1-Lifeact plasmid DNA was transfected using a modified Lipofectamine- based (Invitrogen) protocol. DNA–lipid complexes were generated in serum-free media as described by the manufacturer and used to resuspend a C3H10T1/2 cell pellet containing 3.5 × 105 cells that had been previously washed with serum-free DME. After 10 min of incubation at room temperature, the cell suspension was transferred into a well of a 6-well plate containing 2 ml warm normal growth medium. Fresh medium containing 1 mg/ml Geneticin (Invitrogen) was added after 24 h. Fluorescence time-lapse microscopy and quantification of cell eccentricity. To perform fluorescence time lapse experiments, Lifeact-eGFP transfected C3H/10T1/2 cells were seeded into 6-well plate dishes at a density of 3.5 × 105 cells/well in normal growth medium 48 h before the experiment. The plates were transferred into the heated chamber of an Olympus IX81 motorized inverted microscope (Olympus Medical) and incubated at 37°C throughout the experiment. Pictures of three different fields of view per dish were taken every 10 min for 4 h using an air long working distance 20× objective (NA 0.45). After the capture of the first image, the inflammatory cytokines IL-1β (1, 10, or 100 ng/ml) or TNF (10 or 100 ng/ml) or vehicle control (PBS) were added to the cells. Time-lapse images were then analyzed using the 3D imaging software IMARIS to track and quantify cell eccentricity (the ratio of cell length to cell width). Cell length was quantified as the longest side of the cell in the direction of cell elongation. Cell width measurement was taken perpendicularly to the middle of the cell length. For all three fields of view cell eccentricity was measured for at least five cells and the mean cell eccentricity was determined for each field of view. The overall mean cell eccentricity was then plotted per dish for each time point, and normalized to the general mean cell eccentricity at time point 0 h (time point captured before the addition of cytokines or vehicle control). Statistics. Data were plotted and statistically analyzed using Prism (GraphPad Software). Results were expressed as means ± SEM, and significant differences between multiple groups were identified by one-way analysis of variance, followed by Newman-Keuls Multiple Comparison Test. Whenever two groups were compared, Student’s t test was used. P-values < 0.05 were considered significant. Online supplemental material. Video 1 shows the development of an inflammatory response as induced by TNF in a cremaster muscle venule of an aSMA-RFPcherry x Lys-EGFP-ki mouse immunostained in vivo for EC junctions with the nonblocking C390 anti–PECAM-1 mAb and as observed by real-time confocal microscopy in vivo. Video 2 and Videos 3–5 are four examples of abluminal crawling of neutrophils along pericyte processes as observed by live confocal microscopy in vivo in a cremasteric venule of an aSMA-RFPcherry x Lys-EGFP-ki mouse after TNF-stimulation. Videos 2 and 3 are from the same image sequence with or without the endothelium being visible, respectively. Video 6 shows a pericyte layer hot spot where multiple neutrophils are migrating through the same gap between adjacent pericytes in a cremasteric postcapillary venule of an αSMA-RFPcherry x Lys-EGFP-ki mouse, as observed by live confocal microscopy in vivo. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111622/DC1. Supplementary Material Supplemental Material
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              Inflammation in stroke and focal cerebral ischemia.

              A growing number of recent investigations have established a critical role for leukocytes in propagating tissue damage after ischemia and reperfusion in stroke. Experimental data obtained from animal models of middle cerebral artery occlusion implicate inflammatory cell adhesion molecules, chemokines, and cytokines in the pathogenesis of this ischemic damage. Data from recent animal and human studies were reviewed to demonstrate that inflammatory events occurring at the blood-endothelium interface of the cerebral capillaries underlie the resultant ischemic tissue damage. After arterial occlusion, the up-regulated expression of cytokines including IL-1, and IL-6 act upon the vascular endothelium to increase the expression of intercellular adhesion molecule-1, P-selectin, and E-selectin, which promote leukocyte adherence and accumulation. Integrins then serve to structurally modify the basal lamina and extracellular matrix. These inflammatory signals then promote leukocyte transmigration across the endothelium and mediate inflammatory cascades leading to further cerebral infarction. Inflammatory interactions that occur at the blood-endothelium interface, involving cytokines, adhesion molecules, chemokines and leukocytes, are critical to the pathogenesis of tissue damage in cerebral infarction. Exploring these pathophysiological mechanisms underlying ischemic tissue damage may direct rational drug design in the therapeutic treatment of stroke.
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                Author and article information

                Contributors
                emmanuel.ampofo@uks.eu
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                16 April 2019
                16 April 2019
                2019
                : 9
                : 6119
                Affiliations
                ISNI 0000 0001 2167 7588, GRID grid.11749.3a, Institute for Clinical & Experimental Surgery, Saarland University, ; 66421 Homburg/Saar, Germany
                Article
                42465
                10.1038/s41598-019-42465-7
                6467883
                30992483
                93c1ca2e-22c2-4d63-a38d-3c584fc2c4a0
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 17 April 2018
                : 5 February 2019
                Funding
                Funded by: HOMFORexzellenz 2016/2017
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