ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration †

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration


Introduction
The development of new treatments for cardiovascular diseases requires the identification of molecules that play specific roles in these pathologies.The endothelium forms a barrier between the vessel lumen and the surrounding tissue and is critical for host immune responses during inflammation and infection.When exposed to inflammatory mediators, such as tumour necrosis factor-a (TNF-a) or lipopolysaccharide (LPS), the quiescent endothelium becomes activated and expresses additional pro-inflammatory factors and cell-specific adhesion molecules that enable the extravasation of circulating leucocytes to sites of inflammation.The initial step involves recruitment of leucocytes to the endothelial surface through the binding of leucocyte-expressed L-selectin and endothelium-expressed E-and P-selectins to carbohydrate moieties on the contacting cell.Firm adhesion is mediated by intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelium, which bind b2 and b1 integrins on leucocytes, respectively. 1 -4Excessive leucocyte accumulation contributes to the development of inflammatory disorders, whereas a deficiency in leucocyte trafficking results in host immunosuppression.
An intermediary component of LPS signalling is integrin-linked kinase (ILK), which mediates LPS-induced NOS-2 and COX-2 expression and NO production in macrophages. 5ILK is a widely distributed pseudokinase located in specialized signalling complexes called focal contacts, and regulates signalling from integrins and growth factor receptors.The ILK carboxyl terminal region contains a kinase-like domain, the integrin-binding domain, and motifs for binding to the actin cytoskeleton via alpha and beta parvin adaptor proteins.The ILK amino terminus binds to PINCH, which interacts with the adapter protein Nck2.Nck2 recruits actin regulatory proteins and can bind tyrosine phosphorylated residues of activated growth factor receptors. 6The formation of the ILK-PINCH-Parvin ternary complex is necessary for ILK function and for its recruitment to focal contacts. 7he ability of ILK to integrate signals from integrins and growth factor receptors suggests an involvement in many biological situations.Activation of ILK induces cell survival and cell proliferation, 8,9 and ILK closely regulates capillary formation and the survival of endothelial and progenitor cells. 10,11The role of ILK in pathological processes has been confirmed in several in vivo models, including cardiac repair, 12 cardiomyopathy, 13,14 tumour angiogenesis, 15 and skeletal muscle regeneration. 16ILK knockout models, both germline 17 and conditional, 16,18 -20 have revealed essential roles in development and adult physiology.Endothelial ILK appears to play a specific role in the development of the coronary vasculature (A.Luque et al., in preparation) confirming the variability of endothelial function between different organs. 21n this study, we investigated the role of ILK in the regulation of LPS-elicited inflammatory responses in endothelial cells (EC).Using immortalized EC lines of lung or coronary origin, we show that ILK is required for LPS-induced endothelial expression of ICAM-1 and VCAM-1.Moreover, LPS-stimulated EC lacking ILK show reduced adhesion of primary lymphocytes and monocytic lines and impaired trans-endothelial migration of lymphocytes.These results propose ILK as a potential target for the design of vascular-specific treatments for bacterial associated inflammation.

Animals and cell isolation
EC were isolated and maintained as described 22 from whole lungs and coronary vasculature of C57/BL6 wild-type animals and the genetically modified ILK lox/lox strain. 17,18Spleen lymphocytes were isolated from C57/BL6 wild-type animals (see Supplementary material online, Methods).The study was approved by 'Comite ´de Bioe ´tica y Bienestar Animal.Instituto de Salud Carlos III' conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Positive endothelial selection with antibody-bound magnetic beads
EC were isolated by positive selection using PECAM-1 or ICAM-2 antibodies bound to magnetic beads (Dynabeads w , Invitrogen) as described previously. 22Cells were further purified by FACS (MoFlo, Beckman Coulter) using standard procedures.

Immortalization of EC
EC lines were established using a modified version from previous reports. 22,23Subconfluent primary cultures were incubated in the presence of polybrene (8 mg/mL, Sigma) with supernatant from packaging cells producing recombinant retroviruses that transduced polyoma middle T antigen (virus concentration about 10 -200 pfu/cell).Cells were subjected to a second round of infection.Non-infected cells died within few passages.Table 1 summarizes specific procedures performed.

Flow cytometry
Flow cytometry was performed following standard protocols (see Supplementary material online, Methods).As required, EC transfected with siRNAs or shRNA were incubated with TNF-a (PeproTech), Poly I:C (InvivoGen), or LPS (E. coli 0111:B4 LPS, InvivoGen).Concentrations used are specified for each figure.

Immunofluorescence
Cell immunostaining was performed as described 24 and analysed with a Leica TCS SP5 Multiphoton Confocal microscope.

LDL uptake
Alexa Fluor 488 acetylated low-density lipoprotein (Ac-LDL, L-23380, Molecular Probes) was incubated with EC following manufacturer's recommendation and incorporation was evaluated by flow cytometry.

Western blotting
Endothelial activation and protein expression were monitored by standard immunoblot analysis of downstream phosphorylated targets (see Supplementary material online, Table S1). 24Bands were quantified with Quantity One software (BioRad).

Wound healing assay
Denuded areas ('wounds') were made in endothelial monolayers by scraping with a plastic pipette tip.Plates were then washed and cells stimulated with VEGF (500 ng/mL, Peprotech).Cell migration into the denudated area was monitored by time-lapse photomicroscopy and quantified with

Table 1
Timetable of procedures for the generation of immortalized endothelial lines 2.9 ILK silencing ILK expression was suppressed by two rounds of transient transfection with ILK siRNA (Ambion) using Lipofectamine 2000 (Invitrogen).Alternatively, ILK-silenced EC were generated by the transfection with shRNA plasmids followed by neomycin selection of positively transfected cells (KM05291N, SABiosciences).

Real-time pcr
Transcript expression of adhesion receptors was measured by the SyBr Green method with an ABI Prism 7900 HT Sequence Detection system (see Supplementary material online, Methods). 25

Leucocyte-endothelium co-adhesion assay
Wild-type or ILK-deficient endothelial monolayers in 96 well plates were stimulated with TNF-a, Poly I:C, or LPS for 6 h.EC were then washed several times and CFSE-(10 mM/20 min C1157, Molecular probes)labelled mouse monocyte/macrophage J774 cells, human T cell line Jurkat or spleen lymphocytes were added (125.000-250.000cells/well).After 1 h, wells were washed and fluorescently-labelled cells attached were measured after detergent treatment with a spectrofluorometer plate reader (485 nm/535 nm, Fluoroskan Ascent, Thermo Labsystems).Alternatively, wells were fixed in 3% paraformaldehyde and evaluated by fluorescence microscopy.Each condition was performed in triplicate and adhesion was calculated as a percentage of the adhesion to fibronectin (10 mg/mL, Sigma).Identification of adhesion molecules involved was assessed by including blocking antibodies (see Supplementary material online, Table S1).

Trans-endothelial migration
shRNA-ILK or shRNA-control transfected EC were cultured to confluency on gelatin-coated polycarbonate filter inserts (5 mm pores, Corning) and either left unstimulated or stimulated with LPS or TNF-a for 6 h.After washing the wells, 3 -5 Â 10 5 CFSE-labelled splenocytes in RPMI media/0.5% BSA were placed in the upper compartment and allowed to migrate for 2 h towards SDF-1 (50 ng/mL, PeproTech) placed in the lower chamber.Transmigrated lymphocytes were quantified by microscopy.Migrated splenocytes were represented as the percentage of the total cellular input.Assays were run in triplicate.

Statistical analysis
All data are means + standard deviations of three independent experiments.Statistical significance was estimated by Student's t-test for unpaired observations, with P , 0.05 considered significant.Analysis was performed with the Prism 3 program (GraphPad Software).

Generation and phenotypic description of novel immortalized mouse EC
To examine the role of ILK in inflammatory response of different vascular beds (A.Luque et al., in preparation), we isolated EC from the mouse lung and coronary vasculatures.Since the number of EC obtained, especially from coronary vessels, was insufficient for the experimentation proposed, we generated immortalized cultures to provide infinite biological material.Procedures for generating cell lines included several rounds of endothelial positive selection and two rounds of infection with the polyoma virus middle T (summarized in Table 1).
Light field microscopy revealed the cobblestone monolayer morphology typical of endothelial cultures (Figure 1A).In all immortalized cell lines, flow cytometry detected expression patterns of the endothelial markers PECAM-1, ICAM-1, and ICAM-2 similar to those seen in primary MLEC.In contrast, only MCoEC-31 and MLEC-04 resembled primary EC regarding VCAM-1 levels (Figure 1B).Unless otherwise stated, data shown in subsequent experiments for an individual cell line are representative of similar results obtained with the other immortalized EC lines.
Endothelial polarity and selective permeability depend on specific intercellular connections. 26Immunocytochemical analysis of confluent MLEC-04 confirmed localization of the endothelial homophilic adhesion protein PECAM-1 at intercellular contacts and of the tight junction molecule ZO-1 at the cell -cell boundaries along lateral cell borders (Figure 1C).

Functional characterization of the mouse endothelial lines
The usefulness of the immortalized cell lines as in vitro models of endothelial processes was evaluated through a series of functional assays.EC lines labelled with Alexa Fluor 488 Ac-LDL were brilliantly fluorescent comparable to primary MLEC, indicating characteristic high endothelial Ac-LDL uptake and metabolism; 27 in contrast, fluorescence intensity of a murine keratinocyte cell line (PB) was only slightly above background (Figure 2A).The four endothelial lines also express the receptor tyrosine kinase VEGFR2 (Figures 2B and 3A), which mediates most of the angiogenic actions of VEGF. 28Stimulation of EC with VEGF induced VEGFR2 autophosphorylation, detected by immunoblotting with the anti-phosphotyrosine antibody PY20 followed by anti-VEGFR2 antibody.Sequential membrane reprobing with phospho-specific antibodies detected VEGF-induced phosphorylation of Akt, GSK-3b, and ERK (Figure 2B).VEGF-stimulated endothelial migration was evaluated in wound healing assays. 29ncreased migration of EC in the presence of VEGF is evident from the pronounced closure of the denuded region at 24 h compared with control (Figure 2C).
The induced expression of adhesion molecules-essential for leucocyte recruitment in response to inflammatory stimulation-was tested by activating quiescent EC with LPS or TNF-a.Upon activation, immortalized cultures overexpressed E-selectin, VCAM-1, and ICAM-1 similarly as occurred in primary MLEC.As expected, ICAM-2 and PECAM-1 expression was invariable under these conditions (Figure 2D).Together these results confirm the immortalized cultures as true EC, thus characterizing a useful resource for vascular research.

Expression of the ILK signalosome by EC lines and ILK down-regulation
The four endothelial lines expressed similar levels of ILK protein and its adaptor proteins PINCH-1 and aand b-parvin (Figure 3A), suggesting normal signalling through ILK in these cell types.
The function of ILK in endothelial biology was evaluated by knockdown approaches.ILK expression in all four EC lines was suppressed with siRNA reagents.Approximately 65 -75% of ILK protein was eliminated after two rounds of transfection (Figure 3B).In addition, we used shRNA reagents to generate stable ILK-deficient lines.After shRNA-EC transfection and neomycin selection, ILK expression was reduced to around 25% compared with the shRNA control (Figure 3C).In another approach, MLEC-12 derived from ILK lox/lox animals were positively tested for ILK elimination after cre enzyme expression (Supplementary material online, Figure S1).However, since siRNA technology is applicable to all four EC lines and shRNA EC show similar reductions in ILK expression, siRNA/shRNA approaches were used in all subsequent experiments.

ILK regulates LPS-induced transcription of ICAM-1 and VCAM-1 in EC
To test the role of endothelial ILK in LPS-induced responses, we first analysed the effect of ILK knockdown on the expression of inflammation-related genes.LPS stimulation of MLEC-04 increased the transcription rate of E-selectin by three-fold, and ICAM-1 and VCAM-1 by about 10-and 17-fold.Transfection with ILK-siRNA weakened LPS-induced ICAM-1 and VCAM-1 expression to 60 and 20% of the control values but did not affect LPS-induced expression of E-selectin.ICAM2 was examined as a non-varying endothelial marker (Figure 4A).We next evaluated ILK function in response to different pro-inflammatory conditions.Increasing amounts of LPS (10-500 ng/mL) induced ICAM-1 expression in a concentrationdependent manner, and suppression of this expression in ILK-siRNA-transfected MLEC-04 was significant at higher LPS concentrations (100 and 500 ng/mL).In contrast, ILK knockdown did not significantly interfere with EC expression of ICAM-1 mRNA induced by TNF-a or Poly I:C (Figure 4B and see Supplementary material online, Figure S2).
Further flow cytometry analysis of MCoEC-25 (Figure 5C and D) and MLEC-04 (data not shown) confirmed that the reduced VCAM-1 and ICAM-1 expression in LPS-treated ILK-deficient EC correlates with lower cell surface localization.In contrast, VCAM-1 and ICAM-1 over-expression induced by TNF-a and Poly I:C was not affected by the absence of endothelial ILK.ICAM-2 expression was invariable under all conditions (data not shown).

Down-regulation of endothelial ILK reduces the adhesion of leucocytes to LPS-activated endothelium
ILK-mediated regulation of LPS-induced cell adhesion molecules suggests a regulatory action for ILK on leucocyte adhesion to endothelium, event that was investigated with functional co-adhesion experiments.CFSE labelled J774 cells were added to resting or stimulated siRNA transfected EC and cell attachment was evaluated by fluorescence microscopy (Figure 6A, left panel).LPS and TNF-a treatments increased monocyte adhesion to MLEC-04 monolayers.Although endothelial ILK knockdown did not affect co-adhesion under TNF-a stimulation or resting conditions, it severely inhibited LPS (500 ng/mL)-stimulated adhesion.As it was expected, no ILK regulation occurred at 25 ng/mL because very low EC activation was achieved in this condition (Figure 4B and data not shown).Parallel experiments were quantified by spectrofluorometry of lysed cells (Figure 6A, right panel).
To identify the molecule pairs involved in J774-endothelium binding, first we tested adhesion molecules expressed by the monocytic line.J774 cells express CD11a, CD11b, and CD11c associated with integrin b2, which are receptors for ICAM-1, whereas CD49d associated with b1 integrin, the receptor for VCAM-1.CD45 was examined as leucocytic positive control (Figure 6B).Next we conducted co-adhesion experiments in the presence of blocking antibodies to adhesion components (see manufacturer's datasheet, Supplementary material online, Table S1).Anti-b2 integrin antibody partially reduced adhesion of J774 cells to resting MLEC-04, and severely reduced the adhesion to LPS-activated endothelium (Figure 6C).Thus the main adhesive complex in this experimental setting appears to be endothelial ICAM-1, upregulated in response to LPS, and b2 integrins on J774 cells.
To mimic a more physiological situation, next we used mouse spleen lymphocytes.Adhesion of CFSE-splenocytes to MCoEC-25 (shRNA-Con) was increased after LPS, Poly I:C, or TNF-a endothelial activation.As before, endothelial ILK knockdown (shRNA-ILK) did not affect co-adhesion involving resting EC or stimulated with Poly I:C or TNF-a, but significantly inhibited LPS-stimulated adhesion (Figure 6D).Similar results were obtained with Jurkat cells (see Supplementary material online, Figure S3).

Endothelial ILK regulates extravasation of lymphocytes through LPS-activated endothelium
An effective inflammatory response involves leucocytes attachment to activated endothelium and trans-endothelial migration to the inflamed focus.We therefore next evaluated the role of endothelial ILK in extravasation in transwell migration assays.
shRNA transfected MCoEC-25 (control and ILK) were cultured to confluence on polycarbonate filter and either left unstimulated or stimulated with LPS or TNF-a.After washing the wells, CFSE-splenocytes were placed in the upper compartment and allowed to migrate towards SDF-1 gradient.LPS and TNF-a treatments increased lymphocyte trans-endothelial migration, and were stimulated with LPS (500 ng/mL) for 6 h and expression of selected genes was evaluated by Real-Time PCR.r, resting cells; L, LPS-stimulated cells.(B) MLEC-04 transfected with ILK (I) or control (C) siRNAs were incubated with indicated stimuli for 6 h and expression of ICAM-1 gene was evaluated as before.Data are means + SEM of three independent experiments conducted in triplicate.**P , 0.01 with respect to the control condition.
whereas endothelial ILK knockdown did not affect extravasation across TNF-a stimulated or resting EC, it significantly inhibited LPS-stimulated trans-endothelial migration (Figure 6E).

Discussion
EC play a critical role in regulating inflammation through their capacity to direct leucocyte traffic via the controlled expression of homeostatic and inflammatory mediators. 1A consequence of the stepwise procedure of leucocyte recruitment is that blockade of any step will interrupt the extravasation process and prevent leucocytes entering the surrounding tissue.Therefore, anti-inflammatory strategies to inhibit the function of cell adhesion molecules involved in leucocyte-endothelium interaction are a promising means of regulating the cellular response in inflammation.Our results show that ILK regulates the inflammatory reaction triggered by LPS in four new mouse immortalized EC lines.These EC express ILK and the co-adaptor molecules PINCH and parvin, suggesting normal ILK signalling.LPS induced transcription of the inflammatory genes E-selectin, ICAM-1, and VCAM-1.LPS signals through ILK, as shown by down-regulation of ICAM-1 and VCAM-1 expression in LPS-activated ILK-deficient cells.Moreover, suppression of adhesion molecule expression impaired both the adhesion of monocytic cells and lymphocytes to LPS-activated endothelium and subsequent transendothelial migration.In our system, ILK seems to specifically regulate LPS-triggered EC activation, since the endothelial responses induced by TNF-a or Poly I:C were unaffected.This finding is particularly relevant because endothelial TLR4, the LPS receptor, has been identified as the primary intravascular bacterial sensor, being entirely sufficient to recruit neutrophils to peripheral tissues. 30These findings thus identify a novel regulatory function of endothelial ILK in LPS-induced leucocyte recruitment, suggesting ILK as a potential target for therapeutic modulation of inflammatory disorders triggered by infection with Gram-negative bacteria.
We compared EC cultures derived from lung and coronary circulation because our unpublished results with conditional knockouts indicate that ILK makes a distinct contribution to the endothelium of coronary vessels during development (A.Luque et al., in preparation).However, the experiments presented here revealed no differences between adult vascular beds in the ILK-mediated regulation of LPS triggered responses.Nonetheless, all four endothelial lines were comparable to freshly isolated EC for a range of endothelial phenotypes, as listed in the Supplementary material online, Table S2, positively identifying them as true EC. 31 In addition to data presented in this study, all four cultures express endothelial NOS constitutively and NOS-2 upon LPS activation (Supplementary material online, Figure S4).These well-defined EC lines provide homogeneous cell systems for the reproducible study of physiological and pathological processes including inflammation, vascular tone, blood coagulation, angiogenesis, haemostasis, and transport across blood vessels.
Crosstalk between ILK and FAK signalling has recently been described in the regulation of vascular smooth muscle cells and cardiac maintenance. 13,32FAK regulates ICAM-1 expression upon b1 integrin adhesion, influencing cell viability and maturation. 33nother focal contact protein, Pyk2, mediates up-regulation of LPS-induced VCAM-1 in smooth muscle cells. 34The fact that ILK knockdown did not fully block ICAM-1 and VCAM-1 up-regulation in LPS-treated EC might indicate the activity of compensatory mechanisms arising from crosstalk among different pathways located at the focal contact area, a phenomenon that remains to be investigated.
Another possible explanation for the incomplete suppression of LPS-induced adhesion molecule expression includes the deficient elimination of ILK protein with the knockdown strategies used.The remnant 25 -35% ILK protein expression might reflect limited efficiency of the reagents used or the long half life of the protein. 35he use of ILK knockout cells was not considered because they have low survival and proliferation rates. 17 -19Cell counts indicate that cell proliferation and survival in our immortalized cell cultures were not affected by ILK-siRNA transfection or ILK-shRNA expression (data not shown).Another possibility is that only part of the signalling triggered by LPS in EC is dependent on ILK: the moderate effect of ILK suppression on adhesion molecule expression might reflect complexity of LPS-induced signalling, involving several pathways (ERK, JNK, p38, PI3K/Akt, and NF-kB). 2 Several EC responses to LPS in vitro are similar to events occurring during sepsis, including the production of pro-inflammatory cytokines and the increased surface expression of E-selectin, ICAM-1, and VCAM-1. 3,4Co-adhesion experiments are a widely used in vitro model for studying the regulation of interactions between lymphoid cells and the endothelial layer. 34,36,37The invariable attachment in the presence of conditioned media derived from LPS-activated EC (data not shown) did not motivate us to analyse the regulation of endothelial cytokine production by ILK in this system.
The regulation of LPS-induced ICAM-1 and VCAM-1 overexpression by ILK, but not of E-selectin, suggests a role for ILK in firm adhesion of lymphoid cells to the inflamed endothelium, whereas rolling of circulating cells would be unaffected.Co-adhesion assays in the presence of blocking antibodies revealed that of the molecules tested only b2 integrins mediate J774 attachment to endothelium.Nonetheless, the incomplete suppression of adhesion suggests that other adhesion couples are involved in this process.
To our knowledge, this study provides the first description of the involvement of ILK in the endothelial expression of cell adhesion molecules in response to LPS.The impact of ILK suppression on lymphoid adhesion and subsequent transmigration suggests that vascular ILK could be targeted to moderate the inflammatory response, possibly improving the prognosis of LPS-mediated inflammatory diseases.A previous report showed the involvement of ILK in the expression of SDF-1 and ICAM-1 in EC in hypoxia. 11ILK also regulates the expression of various endothelial markers in an extracellular matrix dependent manner. 37Targeting of ILK has been proposed as a potential anti-cancer approach because of its role in proliferation, migration, and survival of tumour cells. 38The identification of an additional antiadhesive potential for agents that target ILK suggests further applications in the treatment of inflammatory disorders.Several clinical trials targeting cell adhesion molecules are in progress, based on the ability of agents such as pharmacological drugs, antibodies, antagonist peptides, and antisense nucleotides to reduce injury in animal models of inflammatory and immune disease. 39However, in some cases systemic treatment with these agents has shown no therapeutic effect or has adverse outcome.There is therefore a need to redirect strategies to target the cells specifically involved in the disease.Much work has been done to map different endothelium niches, 40 but further research is needed to identify the specific markers expressed during pathological events.

Figure 2
Figure 2 Functional characterization of mouse endothelial lines.(A) AcLDL uptake.EC were incubated with Alexa Fluor 488-AcLDL (dark histograms) and examined by flow cytometry.PB cells were used as control.(B) VEGF signalling.Resting (2) or VEGF-stimulated (þ) MLEC-12 were analysed by western blot with sequential antibody stripping and reprobing of the same membrane.Protein levels are quantified below each blot as a fold-increase relative to b-actin densities.(C) Wound healing assays.Left-hand panels show light field images of scraped VEGF-stimulated or control MCoEC-31 monolayers at 0 and 24 h.Scale bar equals 100 mm.Quantification (right panel) shows surface invaded ¼ a.u.(0 h) 2 a.u.(24 h).(D) Inflammatory response of EC.MCoEC-25, MLEC-04, and primary MLEC were left untreated (white histograms) or stimulated (dark histograms) with LPS (500 ng/mL) or TNF-a (15 ng/mL) for 6 h and expression of surface adhesion molecules was assessed by flow cytometry.'Control' shows isotype matched negative controls.

Figure 3
Figure 3 Expression of the ILK signalosome by EC lines and ILK down-regulation approaches.(A) MLEC-04 (L04), MCoEC-25 (Co25), MCoEC-31 (Co31), and MLEC-12 (L12) express ILK and the adaptor proteins PINCH1 and aand b-parvins as shown western blot of whole cell lysates.(B) Down-regulation of ILK by siRNA.MCoEC-25 were subjected to two rounds of transfection with ILK or control siRNA, and ILK levels were evaluated by western blot.(C) MCoEC-25 and MLEC-04 transfected with ILK-shRNA (I) reduced ILK protein expression compared with control-shRNA (C).In all cases b-actin was assessed as loading control and the protein levels are quantified below each blot as a fold-increase relative to the correspondent b-actin densities.

Figure 5
Figure 5 LPS-induced VCAM-1 and ICAM-1 protein expression is partially inhibited in ILK-deficient EC. (A, B) MCoEC-25 transfected with ILK siRNA (I, ILK) or control siRNA (C, 4611) were left resting (r) or treated with LPS (L, 500 ng/mL) for 6 h.The representative western blots show expression of VCAM-1 (A) and ICAM-1 (B), with b-actin detected as a loading control.The histograms show semi-quantification of relative band densities from four experiments.(C, D) Flow cytometry profiles of VCAM-1 and ICAM-1 on ILK-silenced (I) or control (C) shRNA transfected MCoEC-25 on resting conditions or stimulated for 6 h with LPS (100 and 500 ng/mL), Poly I:C (30 mg/mL), or TNF-a (15 ng/mL).Data are the mean fluorescence intensities of a representative experiment of three performed.

Figure 6
Figure 6 Down-regulation of endothelial ILK reduces adhesion and extravasation of lymphocytes and monocytes to LPS-activated endothelium.(A) Left panels: Fluorescence microscopy showing CFSE-J774 cells attached to resting or activated MLEC-04 cells transfected with control or ILK siRNA; EC were activated before co-attachment for 6 h with LPS (25, 500 ng/mL) or TNF-a (15 ng/mL).Scale bar, 50 mm.Right panel: Spectrophotometric quantification of CFSE-J774 fluorescence in parallel experiments.Results are the means + SEM of four experiments.(B) Summary of cell adhesion molecule expression on J774 cells analysed by flow cytometry.'%', percentage of positive cells; 'Mean', mean fluorescence intensity.Control indicates to isotype matched negative controls.(C) CFSE-J774 cells were incubated with resting or LPS-treated MLEC-04 in the absence (control, solid bars) or presence of integrin b2 subunit blocking antibody (anti-beta2, clear bars).Results are the means + SEM of three experiments.(D) Quantification of the fluorescence signal from CFSE-spleen lymphocytes adhered to resting, LPS (500 ng/mL), Poly I:C (30 mg/mL), or TNF-a (15 ng/mL)-activated MCoEC-25 expressing control-shRNA (solid bars) or ILK-shRNA (clear bars).Results are the means + SEM of three experiments.(E) Splenocyte trans-migration towards SDF-1 across control (C) or ILK (I) shRNA-transfected MCoEC-25 monolayers resting or previously activated with LPS (100, 500 ng/mL) or TNF-a (15 ng/mL).Results are the means + SEM of three experiments.*P , 0.05 and **P , 0.01 with respect to the control.