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Knockdown of mouse VCAM-1 by vector-based siRNA
Transplant Immunology 16 (2006) 185 – 193 www.elsevier.com/locate/trim
Knockdown of mouse VCAM-1 by vector-based siRNA A.K.M. Shamsul Alam a , Oliver Florey b , Michele Weber a,1 , Radhakrishna G. Pillai a , Cliburn Chan a , Peng H. Tan a , Robert I. Lechler a,2 , Myra O. McClure c , Dorian O. Haskard b , Andrew J.T. George a,⁎ b
a Department of Immunology, Division of Medicine, Imperial College London, Hammersmith campus, London W12 0NN, UK BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College London, Hammersmith campus, London W12 0NN, UK c Jefferiss Research Trust Laboratories, Wright-Fleming Institute, Division of Medicine, Faculty of Medicine, Imperial College London, St Mary's Hospital, Norfolk Place, London W2 1PG, UK
Received 7 July 2006; accepted 1 August 2006
Abstract Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology. © 2006 Elsevier B.V. All rights reserved. Keywords: siRNA; Vector-based siRNA; ER stress; Interferon response; Adhesion molecules; VCAM-1; Leukocyte adhesion; Inflammation; Transplantation
1. Introduction Central to the process of transplantation rejection is the recruitment of leukocytes to the site of rejection through the vascular endothelium. This is necessary for both chronic and acute rejection, and can also be a factor in damage caused by reperfusion injury. The recruitment of leukocytes is a multistage process, involving capture and rolling due to interactions between selectins and selectin ligands, activation of the leukocytes ⁎ Corresponding author. Tel.: +44 20 8383 1475; fax: +44 20 8383 2788. E-mail address: [email protected] (A.J.T. George). 1 Current address: Lymphocyte Interaction Laboratory, Cancer Research UK, Lincoln's Inn Fields, London WC2 3PX, UK. 2 Current address: Division of Immunology, Kings College London, Guy's Hospital, London SE1 9RT, UK. 0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2006.08.004
(for example by binding of chemokines to their receptors), firm adhesion mediated by integrin molecules binding to members of the immunoglobulin superfamily (such as ICAM-1 and VCAM-1) and, finally, diapedesis through the endothelium into the tissue [1]. Very Late Antigen 4 (VLA-4)–VCAM-1 interactions may also mediate leukocyte capture and arrest independent of selectins [2]. In the context of transplantation a central interaction is that between VLA-4 and VCAM-1. VCAM-1 is expressed at low levels on endothelial cells, but its expression is upregulated by inflammatory cytokines [3,4]. It is also expressed on other cells including haematopoietic progenitor cells and B cells and has been implicated in T cell co-stimulation and embryonic development [5]. The expression of VCAM-1 on endothelium is upregulated in a wide range of inflammatory conditions, including acute and chronic rejection of transplants [6].
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The importance of VCAM-1–VLA-4 interactions in transplantation has been shown in a variety of animal models [7,8]. Thus, blockade of this interaction by monoclonal antibodies led to prolonged allograft survival in a mouse islet transplantation model [9]. In a rat skin and intestinal allograft model, injection of anti-VLA-4 antibody significantly prolonged graft survival [10]. An alternative to blocking antibodies is to inhibit expression of VCAM-1 on endothelial cells by using anti-sense oligodeoxynucleotides (ODNs), ribozymes, targeted gene disruption, intrabodies and by RNA interference [11–14]. In the experimental setting, the use of VCAM-1 gene knockouts is problematic as it is embryologically lethal in mice [15], though it might be possible to generate conditional knockouts. We describe the use of RNA interference to block expression of VCAM-1. The introduction of double stranded RNA (dsRNA) has been widely used to degrade specific mRNA species, and so ‘knock out’ gene expression [16]. In mammalian cells the introduction of long dsRNA provokes a strong interferon (type 1) response [17]. This can be overcome by the use of short interfering RNA (siRNA) duplexes of 21–23 nucleotides. These can be administered either as synthetic siRNA duplexes, whose effect is transitory [18], or by use of expression vectors. The expression based vectors contain a small DNA insert encoding a short hairpin RNA targeting the gene of interest. Following transcription the short hairpin RNA is rapidly processed into 19–22 nt double stranded RNA. This is assembled into an RNA induced silencing complex (RISC) that binds, cleaves and destroys its cognate RNA [19]. We show here that this technology can be used to achieve functional silencing of VCAM-1, but also demonstrate some of the limitations of the approach that need to be taken into account, particularly when considering its therapeutic application. 2. Objective The objective of this study is to achieve functional silencing of VCAM-1 using vector-based siRNA constructs and to explore the limitations of this approach that are of importance when considering its therapeutic applications. 3. Materials and methods 3.1. Cell lines and primary cells The immortalised human embryonic kidney epithelial cell line, HEK293 [20] (ECACC 85120602), was maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine and antibiotics (100 u/ml Penicillin and 100 μg/ml Streptomycin) (GIBCO, Paisley, UK). The BALB/c derived immortalised murine corneal endothelial cell line (MCEC) was a kind gift from Professor J. Niederkorn (UT Southwestern Medical Centre, Dallas, USA) [21]. These cells were maintained in EMEM with EBSS supplemented with 10% FCS, 2 mM L-glutamine, antibiotics and 25 μg/ml Fungizone, 1% sodium pyruvate and 1% MEM vitamin mixture (Cambrex, Haverhill, UK). The subcutaneous haemangioma endothelial cell line, sEnd.1, was derived from a C3H mouse [22]. These cells were maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, antibiotics, 1% sodium pyruvate, 1% MEM vitamin mixture and 1% non-essential amino acids (Invitrogen, Paisley, UK).
Jurkat cells, a transformed human lymphoblastic leukaemia T-cell line [23], were maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine and antibiotics. BALB/c mice primary T cells were purified from spleen cells following incubation with rat anti-mouse class II, anti-CD16/32 and anti-B22 (Caltag, Silverstone, UK) antibodies followed by magnetic separation using sheep antirat beads (Dynal Biotech, Norway). These were maintained overnight in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 5 × 10− 5 M mercaptoethanol (2-ME) and antibiotics.
3.2. Cloning of mouse VCAM-1 gene Whole RNA was extracted from the MCECs using TRIzol® (Life Technologies, UK) according to the manufacturer's instructions. A 5 μg aliquot of total RNA was reverse transcribed into cDNA using oligo dT primer (2.5 mM), 10 mM dNTPs, RNAse inhibitor and reverse transcriptase (GIBCO-BRL, UK). The mouse VCAM-1 gene was PCR-amplified using the primers, 5′-CTCCA CATCTCGAGATGCCTGTGAAGATGGTCG-3′ and 5′-CATTTCAGAATT CAGCTACACTTTGGATTTCTGT-3′ containing Xho1 and EcoR1 restriction sequences at 5′ and 3′ ends, respectively. The gene was cloned into the pcDNA3.1 (−) plasmid (Invitrogen), and the sequence checked. This construct was termed pcDNA3.1/mVCAM-1 (abbreviated in figures to V3).
3.3. Selection and cloning of mouse VCAM-1-specific sequences for vector-based RNA interference Nine target sequences were selected from the published mouse VCAM-1 mRNA sequences (both the coding region and the 3′ non-coding region) (NCBI database accession no. BC029823). Each sequence was 19 nucleotides length (Table 1). The sequences were chosen so that the GC content was 30–50%, and there was no sequence of four or more adenines or thymidines. The sequences were blast-searched against murine EST libraries to ensure that they were not homologous to other sequences. Forward and reverse oligonucleotide primers were designed as described [19] with a nine nucleotide spacer sequence, flanked by BglII and HindIII sequences at the 5′ and 3′ ends respectively. These were annealed and cloned in to the pSUPER vector (Oligoengine, USA) between the BglII and HindIII sites. The negative controls (NC1, NC5, NC19) are specific for Hepatitis C virus mRNA. All constructs were sequenced to confirm correct insertion of the oligonucleotides. For stable transfection, the oligonucleotides encoding si12, si14 and si16 and NC1 were cloned into pSilencer.puro (Ambion Inc., USA). For some experiments an intracellular antibody (E6) consisting of a single chain Fv fused to the KDEL sequence cloned into the pEF/myc/ER plasmid (Invitrogen, USA) was used (manuscript in preparation).
3.4. Transfection Lipofection was carried out using LipofectAmine (Life Technologies, UK) following the manufacturer's protocol optimized for different cell types. For stable transfection the sEnd.1 cell line was transfected with pSilencer.puro vector-based siRNA constructs, selected with Puromycin (4 μg/ml) and cloned by limiting dilution. The sEND.1 cells, and clones derived from them were induced to express surface VCAM-1 molecule by stimulating with 50 ng/ml TNFα for 24 h.
3.5. Flow cytometry Flow cytometric analysis was carried out on cells stained with monoclonal antibodies against mouse VCAM-1, CD40 and PDL1 (all from e-Bioscience, California, USA). FITC-conjugated rabbit anti-rat Ig was used as secondary antibody (Sigma, UK).
3.6. Western blotting Cell lysates were prepared and Western blotted, as described [24]. Blots were probed with monoclonal antibodies against mouse β-actin (Santa Cruz) and VCAM-1.
Sequence name
Starts at
si4 sense si4 antisense si5 sense si5 antisense si8 sense si8 antisense si12 sense si12 antisense si14 sense si14 antisense si15 sense si15 antisense si16 sense si16 antisense si19 sense si19 antisense si23 sense si23 antisense
672 967 1113 1198 1231 1639 2004 2213 2764
Forward primer sequence
Spacer sequence
Reverse primer sequence
5′-GATCCCCGGATCCAGAGATTCAATTCTTCAAGAGAGAATTGAATCTCTGGATCCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGGATCCAGAGATTCAATTCTCTCTTGAAGAATTGAATCTCTGGATCCGGG-3′ 5′-GATCCCCGAACTACAAGTCTACATCTTTCAAGAGAAGATGTAGACTTGTAGTTCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGAACTACAAGTCTACATCTTCTCTTGAAAGATGTAGACTTGTAGTTCGGG-3′ 5′-GATCCCCTGAAGTTCTCCAGCTTCTCTTCAAGAGAGAGAAGCTGGAGAACTTCATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATGAAGTTCTCCAGCTTCTCTCTCTTGAAGAGAAGCTGGAGAACTTCAGGG-3′ 5′-GATCCCCGGAGTTAATCTGATTGGGATTCAAGAGATCCCAATCAGATTAACTCCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGGAGTTAATCTGATTGGGATCTCTTGAATCCCAATCAGATTAACTCCGGG-3′ 5′-GATCCCCGTGGAATTAGTTGTTCAAGTTCAAGAGACTTGAACAACTAATTCCACTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGTGGAATTAGTTGTTCAAGTCTCTTGAACTTGAACAACTAATTCCACGGG-3′ 5′-GATCCCCTTACTGAAGGGGGAGACTATTCAAGAGATAGTCTCCCCCTTCAGTAATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATTACTGAAGGGGGAGACTATCTCTTGAATAGTCTCCCCCTTCAGTAAGGG-3′ 5′-GATCCCCTACTACACTCACCTTCATGTTCAAGAGACATGAAGGTGAGTGTAGTATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATACTACACTCACCTTCATGTCTCTTGAACATGAAGGTGAGTGTAGTAGGG-3′ 5′-GATCCCCCATGGATAATCCTGAAGAATTCAAGAGATTCTTCAGGATTATCCATGTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAACATGGATAATCCTGAAGAATCTCTTGAATTCTTCAGGATTATCCATGGGG-3′ 5′-GATCCCCTGGTCCCATGATGTGTATGTTCAAGAGACATACACATCATGGGACCATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATGGTCCCATGATGTGTATGTCTCTTGAACATACACATCATGGGACCAGGG-3′
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Table 1 siRNA primer sequences selected from mouse VCAM-1 mRNA
The table shows both the sense and antisense oligonucleotides used to make the siRNA constructs. The sequences encoding the forward and reverse sequences for the siRNA are shown, between these is a short sequence making a hairpin loop. The starting nucleotide in the VCAM-1 cDNA sequence is indicated (the VCAM-1 coding region sequences is between 322 and 2541). All of the target sequences are from the coding region except for si23 which has been designed from the 3′ UTR.
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3.7. RT-PCR and real-time PCR Total RNA was extracted using Trizol RNA extraction kit (Invitrogen) from 2– 5 × 106 cells. Total (5 μg) RNA was used for reverse transcription in a final volume of 40 μl. One μl of cDNA was used for semi-quantitative and real-time RT-PCR amplifications. Semi-quantitative RT-PCR amplification was performed for VCAM-1, UPR and ds-RNA responsive genes using the following primers; VCAM-1 sense 5′-ATGACATGCTTGAGCCAGG-3′, antisense 5′-GTGTCTCC TTCTTTGACACT-3′; heavy chain binding protein (BiP) sense 5′-CTGGGTACA TTTGATCTGACTGG-3′, antisense 5′-GCATCCTGGTGGCTTTCCAGCCA TTC-3′; C/EBP homologous protein (CHOP) sense 5′-GTCCAGCTGGGAGCT GGAAG-3′, antisense 5′-CTGGTCAGGCGCTCGATTTCC-3′; growth arrest and DNA damage-inducible protein 45 (GADD45) sense 5′-GAGCAGAAGA CCGAAAGGATG-3′, antisense 5′-CTTCAGTGCAATTTGGTTCAG-3′; Stanniocalcin 2 (STC2) 5′-AGGAGAACGTCGGTGTGATT-3′, antisense 5′CTGTTCACACTGAGCCTGGA-3′; activating transcription factor 4 (ATF4) sense 5′-TCCTGAACAGCGAAGTGTTG-3′, antisense 5′-ACCCATGAGGT TTCAAGTGC-3′; 2 5 oligoadenylate synthetase 1 (OAS1) sense 5′-AGGTGGTA AAGGGTGGCTCC-3′, antisense 5′-ACAACCAGGTCAGCGTCAGAT-3′. Real-time PCR amplification was performed in a lightcycler (Roche) using SYBR green master mix (Sigma-Aldrich Company Limited, Poole, UK) according to the manufacturer's instructions. The PCR protocol included an initial denaturation step (95 °C, 10 min) followed by 40 amplification and quantification cycles (95 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s and a single acquisition at 81 °C), and a melting curve program (65–95 °C). Three sets of primers were used, each set spanning the corresponding sequences selected for that siRNA to give a PCR product of about 200 bp. The primers used were: si12 primers; sense (5′-AGTTCTCCAGCTTCTCTCAGGA-3′), antisense (5′GGGAGATGTCAACAATAAATGG-3′); si14 primers; sense (5′-GCCATCCT CACCTTAATTGCTA-3′), antisense (5′-ATAGCAGCACACGTCAGAA CAA-3′); si16 primers; sense (5′-ATGGGATACCAGCTCCCAAA-3′), antisense (5′-TGCTAATTCCAGCCTCGTTAAT-3′). The relative mRNA expression level was calculated using the standard curve of pcDNA3.1/mVCAM-1.
3.8. Static adhesion assay sEnd.1 cells, either untransfected or transfected with the pSUPER/si plasmid constructs, were seeded in 96-well plates at 1.25 × 104 cells/well and grown to confluence (48 h). Cells were either unstimulated or stimulated with TNFα (50 ng/ml) for 24 h before the assay. Jurkat cells (1–2 × 107) were labelled with Calcein-AM (Molecular probes, UK) at 10 μg/ml for 1 h. After washing with warm PBS, 2.5 × 105 Jurkat cells were added per well for 30 min at 37 °C. Unbound Jurkat cells were gently washed off with three changes of warm PBS, and the fluorescence was measured in a plate reader. Each experiment was performed in quadruplicate.
3.9. Flow adhesion assay The flow chamber adhesion assay was modified from a published method [25]. Briefly, untransfected and stably transfected sEnd.1 clones were grown for 48 h to confluence in 9-cm2 Nunc Slide Flaskettes (Nalge-Nunc International, Roskilde, Denmark), unstimulated or stimulated with TNFα (50 ng/ml) for 24 h before the assay. These were mounted in a parallel plate flow chamber (channel height 0.15 cm). Mouse primary T cells were either unstimulated or stimulated with 10 μg/ml of Con A for 3 h. Jurkat cells or primary T cells were labelled with Calcein-AM as described above, resuspended at 0.25 × 106 cells/ml in RPMI 1640/2% FCS (viscosity 0.007 P) and perfused at 37 °C over the monolayers at a shear stress of 1.5 dyn/cm2 for the Jurkat cells. For the primary T cells, the shear stress used was 1.0 dyn/cm2 for 1 min to allow the cells to cover the monolayer and then 0.75 dyn/cm2 for the rest of the experiment. Experiments were visualized using an inverted Nikon Diaphot 300 fluorescence microscope connected to a colour video camera. Recording commenced after 2 min of perfusion and consisted of a 15 s recording of 10 random fields using a 10× objective; the area of the analysed field was 800 μm × 400 μm. Each experiment was repeated at least three times on separate days.
Analysis was performed using EML Motion Analysis software (Ed Marcus Laboratories, Brighton, MA). The following indices were calculated: the minimum and maximum velocity of individual cells, the number of rolling cells per field and the number of arrested cells per field. Rolling cells were defined as those with a mean velocity of less than 100 μm/s. Arrested cells were defined as those moving less than 5 μm in 10 s.
4. Results 4.1. Isolation of siRNA constructs specific for VCAM-1 Based on the sequence of murine VCAM-1, we designed nine siRNA constructs (Table 1). Candidate siRNA sequences were generated as described in Materials and methods against the entire VCAM-1 sequence, including the untranslated regions, and the nine constructs were chosen randomly from these. Appropriate oligonucleotides were made and successfully cloned into the pSUPER vector [19,26–29]. To determine whether the siRNA constructs inhibited VCAM-1 expression, we co-transfected them into HEK293 cells together with a construct encoding VCAM-1. All 9 constructs inhibited VCAM-1 expression to, or close to, background levels, with no effect of control siRNA constructs (NC5 and NC19) (data not shown). 4.2. Transfection of corneal endothelial cell lines We then transfected the constructs into the MCEC corneal endothelial cell line, which constitutively expresses VCAM-1. The cells were assessed by flow cytometry for the expression of VCAM-1 after 12, 24, 36 and 48 h. Transfection of MCEC cells using the lipofection approach results in around 50% transfection efficiency when assessed with a plasmid encoding EGFP (data not shown). As is shown in Fig. 1A and B for representative constructs at 36 h, and in Fig. 1E for all the constructs, the majority of the siRNA constructs were effective at inhibiting VCAM-1 expression. The degree of inhibition was not complete, as would be expected given the transfection efficiency. Inhibition was seen at all time points, with a trend to maximal inhibition at 48 h. Some constructs (si15 and si19) did not inhibit VCAM-1 expression. Transfection with control constructs (siNC1, siNC5 and siNC19) did not affect VCAM-1 expression. To determine the specificity of VCAM-1 inhibition, the cells were stained for CD40 expression. There were no alterations in CD40 expression with any of the constructs at any of the time points (Fig. 1F). The three constructs that showed greatest inhibition of VCAM-1 expression (si12, si14 and si16) were transfected into MCECs. After 36 h cell lysates were made and analysed by SDS-PAGE and Western blotting for expression of VCAM-1 protein. There was considerable reduction in VCAM-1 protein, with no reduction in cells transfected with a control construct (Fig. 1G). We also transfected the cells with an intracellular antibody that binds mouse VCAM-1 (E6, manuscript in preparation), and showed a reduction in VCAM-1 expression in these cells. The level of VCAM-1 mRNA was determined by real time RT-PCR in MCECs transfected with the constructs. As shown in Fig. 1H (results of one representative primer set, si12) there was downregulation of mRNA levels for most of the constructs, though as with protein expression this was not seen for si15 and si19. Cells transiently transfected with the E6 intracellular anti-VCAM-1 antibody did not show a reduction in VCAM-1 mRNA (not shown), consistent with the post-translational action of this molecule. 4.3. Transfection of vascular endothelial cell lines The vascular endothelial cell line sEnd.1, which is derived from skin haemangioma cells, expresses very low levels of VCAM-1;
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Fig. 1. Reduction of VCAM-1 expression in MCE cells. Flow cytometric analyses of the expression of VCAM-1 or CD40 at 36 h following either no transfection (Untr.) or transfection with negative control, NC1 (A, C) or two representative VCAM-1 specific constructs si12 and si14 (B, D). The shaded profile shows staining of untransfected MCE cells by isotype control antibody. The histograms (E, F) show the mean fluorescence ration (MFR) of intensity of VCAM-1 or CD40 expression 12, 24, 36 and 48 h following transfection with the nine VCAM-1 specific and 3 negative control (NC1, NC5, NC19) siRNA constructs. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three experiments. (G) Lysates of MCEC untransfected, or transfected with NC5, si12, si14 or si16 siRNA constructs and an anti-porcine VCAM-1 scFv construct (E6) that cross-reacts with mouse VCAM-1 were prepared at 36 h post-transfection, separated by SDS-PAGE, western blotted and probed with an anti-mouse VCAM-1 antibody. β-Actin was used as loading control. Densitometric analysis of the VCAM-1 blot shows a siRNA specific reduction (original western blot in inset). Real-time RT-PCR results (H) show mRNA copy-numbers at 36 h following transient transfection with all of the nine specific siRNA constructs and negative controls (NC1, NC5). Results shown are those obtained using one of the representative primer sets, si12. HPRT was used as internal control. P b 0.05 for all VCAM-1 siRNA constructs when compared to MCEC using ANOVA. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
however, following stimulation with TNFα, VCAM-1 expression is upregulated. Cells were transfected with the constructs and the expression of VCAM-1 was determined 24, 36 and 48 h later. When indicated, the cells were activated by treatment with 50 ng/ml TNFα 24 h prior to analysis. There was upregulation of VCAM-1 expression in sEnd.1 cells that were untransfected or transfected with a control siRNA construct and then stimulated with TNFα. This upregulation of VCAM-1 expression was inhibited by all the siRNA constructs, though
to a varying extent (si8, si15 and si19 being less effective than the other constructs) (Fig. 2). We also assessed expression of Programmed Death Ligand 1 (PDL1). This is expressed at low levels on resting sEnd.1 cells, but is upregulated by cytokines. There was increased PDL1 expression (especially at later time points) in all cells transfected with siRNA constructs. This included cells transfected with the control siNC1 construct, indicating that this is an effect of transfection and/or siRNA
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Fig. 2. VCAM-1 and PDL1 expression by cytokine-stimulated sEnd.1 cells following transient transfection with siRNA constructs. (A) The sEnd.1 cells were either untransfected (Untr) or transfected with siRNA constructs (NC1, si12, si14) and then stimulated immediately with TNFα. 36 h later these were analysed for VCAM-1 expression by flow cytometry. The shaded profiles show staining of untransfected cells by isotype control antibody. The sEnd.1 cells were transfected with siRNA constructs and analysed by flow cytometry for VCAM-1 (B) and PDL1 (C) expression at 24, 36 and 48 h. Cells were stimulated with TNFα 24 h prior to analysis. The MFR of unstimulated cells (sEND-Unst) and untransfected cells stimulated with TNFα (sEND-St) is shown. A marked reduction in VCAM-1 expression is seen with all the VCAM-1 specific constructs (P b 0.05 when compared to sEND-St using ANOVA). There is an increase in PDL1 expression for all siRNA constructs (P b 0.05 when compared to sEnd-St using ANOVA). Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
expression, rather than a specific effect of the VCAM-1 siRNA constructs (Fig. 2C). 4.4. Generation of stable vascular cell lines In order to generate stable cell lines expressing siRNA, the constructs were subcloned into pSilencer.puro plasmid. Stable clones of sEND.1 cells containing these constructs were generated and analysed for expression of VCAM-1 following TNFα stimulation. Data from nine representative clones are shown in Fig. 3A (nomenclature: first number indicates the siRNA construct, and the second the clone number). The majority of clones showed little upregulation of VCAM1 expression following stimulation, when compared to either parental sEnd.1 or clones containing control siRNA constructs. Four stable clones (12.2, 12.3, 12.7 and 14.21) were selected for further analysis. The clones expressed normal levels of ICAM-1 (data not shown), and somewhat upregulated PDL1 expression 24 h after TNFα stimulation (Fig. 3B). Stable transfection of VCAM-1 siRNA did not influence PDL1 expression in unstimulated cells. 4.5. T cell adhesion to siRNA expressing cells To study the functional effect of VCAM-1 knockdown, the ability of the human T cell line, Jurkat, to adhere to a monolayer of sEnd.1 cells was determined using a static assay (Fig. 4A). Adherence of Jurkat to parental sEnd.1 cells is dependent on VCAM-1, as shown using an anti-VCAM-1 blocking antibody 429. Blocking ICAM-1 with antibody YN1 also reduced binding of Jurkat cells. The amount of
adhesion of Jurkat cells to 12.2, 12.3 12.7 and 14.21 clones was greatly reduced. While static assays give useful information about the adhesion of cells to monolayers, they are not representative of the situation in vivo, where leukocyte adherence and transmigration occur in the presence of flow. We, therefore, carried out adhesion assays to endothelial monolayers under flow conditions, analysing both the number of cells rolling and those that have undergone arrest. Few Jurkat cells were seen either rolling or firmly adhered to the parental unstimulated sEnd.1 cells (Fig. 4B). However, following stimulation with cytokines there was an increase in the number of arrested cells. This was inhibited by antiVCAM-1 antibody, 429. The adherence of Jurkat cells to 14.21 (following cytokine stimulation) was greatly reduced. There was little rolling of any cells, consistent with the negligible level of E-selectin expression seen 36 h after stimulation of endothelial cells (data not shown). The above experiments were performed in a xenogeneic combination. We, therefore, repeated them using freshly isolated mouse T cells (Fig. 4C). There was some adherence of the murine cells to unstimulated sEnd.1 cells, which was increased by stimulation. However, with the 14.21 clone there was a decrease in adhesion under both stimulated and unstimulated conditions. Taken together these data indicate that inhibition of VCAM-1 expression by siRNA is capable of inhibiting the VCAM-1 dependent adhesion of T cells to endothelial cells. 4.6. Non-specific activation of endothelial cells by siRNA The increase in PDL1 expression following siRNA transfection suggests that the constructs have a non-specific effect on the cells.
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Fig. 3. VCAM-1 expression in stable sEnd.1 clones. The siRNA sequences (NC1, si12, si14 and si16) were cloned into the pSilencer.puro plasmid and the sEnd.1 cells were either untransfected (Untr.) or transfected with these constructs. Following cloning and selection with Puromycin, the cells were analysed for expression of VCAM-1 (A) or PDL1 (B) either without stimulation (Unst.) or 24 h after stimulation with TNFα. Most of the clones show a dramatic reduction of VCAM-1 expression while there was an increase in PDL1 expression (P b 0.05 for all VCAM-1 siRNA clones when compared to Untr. using ANOVA). Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
Moreover, the sEnd.1 cells stably transfected with the siRNA constructs showed slower growth than untransfected cells (data not shown). In addition, some clones, over time, had a tendency to ‘revert’ with a gradual increase in expression of VCAM-1. These data are consistent with there being a growth disadvantage to cells expressing siRNA. Similar slower growth was also seen with the clones expressing control siRNA. We, therefore, looked for upregulation of stress response molecules. In MCEC cells transiently transfected with the siRNA constructs there was upregulation of BiP, CHOP, GADD45, ATF4 and STC2 as detected by RT-PCR, which was similar whether the cells were transfected with VCAM-1 specific constructs or control constructs (Fig. 5A). However, there was no upregulation of IFNβ following transfection (data not shown). In stable sEND.1 clones there was similar upregulation of the same response elements as well as OAS1 (Fig. 5B).
5. Discussion Interactions between adhesion molecules expressed on endothelium and leukocytes are central to graft rejection, and blocking these interactions can prolong graft survival [6,8,10]. In this study, we sought to determine the effects of reducing VCAM-1 expression using siRNA. We have shown that siRNA can greatly reduce VCAM-1 expression in both corneal endothelial cells
Fig. 4. Static and flow adhesion assay of Jurkat T cells (J6) or mouse primary T cells on sEnd.1 cell monolayers. In all cases except where indicated as unstimulated (Unst.), the endothelial cells and/or the stable clones were stimulated with TNFα 24 h prior to the experiment. (A) sEnd.1 cells were either untransfected (Untr.) or stably transfected with siRNA constructs including the negative control (NC1) grown as monolayers and adherence of Jurkat cells was assessed under static conditions. When indicated, anti-mouse VCAM-1 (429), anti-mouse ICAM1 (YN1) and isotype control antibodies were added. J6 indicates the total fluorescence activity of labelled Jurkat T cells only. Two of the four stable siRNA clones used showed markedly reduced adhesion of Jurkat cells. (B) Jurkat cells were analysed over cytokine activated sEnd.1 endothelial cell monolayers under flow conditions and the number of rolling or arrested cells was determined. There was significantly reduced arrest of Jurkat cells especially on the stable clone 14.21. (C) Primary mouse T cells were also assessed under flow conditions for their rolling and arrest on unstimulated or stimulated sEnd.1 cell monolayers. The stable clone, 14.21 monolayer again showed a statistically significant reduction of arrest of the T cells. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three independent experiments.
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Fig. 5. Stress responses following siRNA transfection. (A) MCEC were either untransfected or transiently transfected with the siRNA constructs including negative controls (NC1, NC5, NC19). Alternatively (B) sEND.1 cells stably transfected with siRNA constructs were used. RT-PCR for stress markers was performed either 36 h after transfection (MCEC) or 24 h after stimulation with TNFα (sEND-1). Where indicated PC (positive control), cells were treated with Thapsigargin (300 nM) for 10 h. Where indicated ‘No RT’ reverse transcriptase was omitted from MCEC reactions. β-Actin was used as loading control.
(that constitutively express VCAM-1) and vascular endothelial cells (that upregulate VCAM-1 upon activation). This blockade of VCAM-1 expression is effective at reducing T cell adhesion in both static and flow assays. RNA interference has been used to knock down gene expression in different organisms, including plants and Caenorhabditis elegans and Drosophila, and is widely used to study gene function [30,31]. In addition it has been used in preclinical models in a therapeutic context, for example to inhibit the expression of Hepatitis B virus [28,32] and HIV mRNA [26,33]. We would envisage that siRNA could be used to block VCAM-1 expression in the context of transplantation using either gene therapy approaches to express the constructs in endothelial cells [11,29] or in xenotransplantation by making transgenic animals. The use of endothelial-specific promoters [34] could restrict expression to the endothelium. However, for siRNA technology to be successful, it is necessary for its action to be specific. In our study, there is a clear effect of siRNA constructs on cell phenotype, with upregulation of the expression of PDL1 in vascular endothelial cells. In addition, cells containing siRNA constructs (both VCAM-1 specific and control) grew more slowly than cells not containing the constructs (even in the absence of selection Puromycin in the medium), while cells transfected with ‘empty’ vector alone grew normally (data not shown), suggesting some non-specific toxic or inhibitory effect of the siRNA constructs. It is recognised that long dsRNA (N 30 nucleotides) induce a strong interferon response through PKR, OAS1 and TLR3 pathways [18,35–37] inhibiting general protein synthesis. It is often assumed that
synthetic 21 nt siRNA can specifically suppress gene expression in mammalian cells, though it has been reported that transfection with such constructs can lead to an interferon response [38–40]. In order to overcome this problem, plasmid vectors using the pol III H1-RNA or U6 gene promoters have been designed to produce smaller 19 nt siRNA transcripts within the cells [19,41]. The use of the U6 promoter has been reported to nonspecifically induce OAS1 due to the formation of longer double stranded RNA molecules [35,42]. In our study, we used a vector with the pol III H1-RNA promoter. We saw no induction of interferon β response or OAS1 gene induction following transient transfection with the plasmids, although we did observe upregulation of the OAS1 gene in stable clones. However, we did observe a stress response, with increased transcription of CHOP, GADD45 and STC2. These can all be upregulated by increases in ATF4, as observed, which is induced by the production of phosphorylated eukaryotic translation initiation factor (eIF-2α). Phosphorylation of eIF-2α can be induced either by the unfolded protein response (via phosphorylation of PKR-like ER-associated kinase (PERK) [43]) or through activation of phosphorylated protein kinase R (PKR) (induced via the dsRNA pathway) [44]. These stress responses results in inhibition of protein synthesis and RNA cleavage (phosphorylated eIF-2α and OAS1) and apoptosis (CHOP and GADD45). In addition they can increase the transcription of a variety of genes (including BiP). These findings can explain the slower growth of the cells expressing siRNA constructs, as well as the alterations in expression of other proteins. RNA interference has potential for the modulation of cellular phenotype with a view to preventing graft rejection. This includes the inhibition of adhesion molecule expression. However, the non-specific upregulation of stress responses and the consequent non-specific effects on cell phenotype highlight the potential problems with this approach. Acknowledgments This work was funded by the MRC and Action Medical Research. Andrew George was a BBSRC Research Development Fellow. PH Tan was an MRC/Royal College of Surgeon's (Edinburgh) Clinical Research Fellow. References [1] Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:827–72. [2] Jones DA, McIntire LV, Smith CW, Picker LJ. A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions. J Clin Invest 1994;94:2443–50. [3] Briscoe DM, Schoen FJ, Rice GE, Bevilacqua MP, Ganz P, Pober JS. Induced expression of endothelial-leukocyte adhesion molecules in human cardiac allografts. Transplantation 1991;51:537–9. [4] Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokineinduced endothelial protein that binds to lymphocytes. Cell 1989;59: 1203–11. [5] Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol 2003;3 (4):292–303. [6] Stegall MD, Ostrowska A, Haynes J, Karrer F, Kam I, Gill RG. Prolongation of islet allograft survival with an antibody to vascular cell adhesion molecule 1. Surgery Aug 1995;118(2):366–9 [discussion 9-70].
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Knockdown of mouse VCAM-1 by vector-based siRNA A.K.M. Shamsul Alam a , Oliver Florey b , Michele Weber a,1 , Radhakrishna G. Pillai a , Cliburn Chan a , Peng H. Tan a , Robert I. Lechler a,2 , Myra O. McClure c , Dorian O. Haskard b , Andrew J.T. George a,⁎ b
a Department of Immunology, Division of Medicine, Imperial College London, Hammersmith campus, London W12 0NN, UK BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College London, Hammersmith campus, London W12 0NN, UK c Jefferiss Research Trust Laboratories, Wright-Fleming Institute, Division of Medicine, Faculty of Medicine, Imperial College London, St Mary's Hospital, Norfolk Place, London W2 1PG, UK
Received 7 July 2006; accepted 1 August 2006
Abstract Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology. © 2006 Elsevier B.V. All rights reserved. Keywords: siRNA; Vector-based siRNA; ER stress; Interferon response; Adhesion molecules; VCAM-1; Leukocyte adhesion; Inflammation; Transplantation
1. Introduction Central to the process of transplantation rejection is the recruitment of leukocytes to the site of rejection through the vascular endothelium. This is necessary for both chronic and acute rejection, and can also be a factor in damage caused by reperfusion injury. The recruitment of leukocytes is a multistage process, involving capture and rolling due to interactions between selectins and selectin ligands, activation of the leukocytes ⁎ Corresponding author. Tel.: +44 20 8383 1475; fax: +44 20 8383 2788. E-mail address: [email protected] (A.J.T. George). 1 Current address: Lymphocyte Interaction Laboratory, Cancer Research UK, Lincoln's Inn Fields, London WC2 3PX, UK. 2 Current address: Division of Immunology, Kings College London, Guy's Hospital, London SE1 9RT, UK. 0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2006.08.004
(for example by binding of chemokines to their receptors), firm adhesion mediated by integrin molecules binding to members of the immunoglobulin superfamily (such as ICAM-1 and VCAM-1) and, finally, diapedesis through the endothelium into the tissue [1]. Very Late Antigen 4 (VLA-4)–VCAM-1 interactions may also mediate leukocyte capture and arrest independent of selectins [2]. In the context of transplantation a central interaction is that between VLA-4 and VCAM-1. VCAM-1 is expressed at low levels on endothelial cells, but its expression is upregulated by inflammatory cytokines [3,4]. It is also expressed on other cells including haematopoietic progenitor cells and B cells and has been implicated in T cell co-stimulation and embryonic development [5]. The expression of VCAM-1 on endothelium is upregulated in a wide range of inflammatory conditions, including acute and chronic rejection of transplants [6].
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The importance of VCAM-1–VLA-4 interactions in transplantation has been shown in a variety of animal models [7,8]. Thus, blockade of this interaction by monoclonal antibodies led to prolonged allograft survival in a mouse islet transplantation model [9]. In a rat skin and intestinal allograft model, injection of anti-VLA-4 antibody significantly prolonged graft survival [10]. An alternative to blocking antibodies is to inhibit expression of VCAM-1 on endothelial cells by using anti-sense oligodeoxynucleotides (ODNs), ribozymes, targeted gene disruption, intrabodies and by RNA interference [11–14]. In the experimental setting, the use of VCAM-1 gene knockouts is problematic as it is embryologically lethal in mice [15], though it might be possible to generate conditional knockouts. We describe the use of RNA interference to block expression of VCAM-1. The introduction of double stranded RNA (dsRNA) has been widely used to degrade specific mRNA species, and so ‘knock out’ gene expression [16]. In mammalian cells the introduction of long dsRNA provokes a strong interferon (type 1) response [17]. This can be overcome by the use of short interfering RNA (siRNA) duplexes of 21–23 nucleotides. These can be administered either as synthetic siRNA duplexes, whose effect is transitory [18], or by use of expression vectors. The expression based vectors contain a small DNA insert encoding a short hairpin RNA targeting the gene of interest. Following transcription the short hairpin RNA is rapidly processed into 19–22 nt double stranded RNA. This is assembled into an RNA induced silencing complex (RISC) that binds, cleaves and destroys its cognate RNA [19]. We show here that this technology can be used to achieve functional silencing of VCAM-1, but also demonstrate some of the limitations of the approach that need to be taken into account, particularly when considering its therapeutic application. 2. Objective The objective of this study is to achieve functional silencing of VCAM-1 using vector-based siRNA constructs and to explore the limitations of this approach that are of importance when considering its therapeutic applications. 3. Materials and methods 3.1. Cell lines and primary cells The immortalised human embryonic kidney epithelial cell line, HEK293 [20] (ECACC 85120602), was maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine and antibiotics (100 u/ml Penicillin and 100 μg/ml Streptomycin) (GIBCO, Paisley, UK). The BALB/c derived immortalised murine corneal endothelial cell line (MCEC) was a kind gift from Professor J. Niederkorn (UT Southwestern Medical Centre, Dallas, USA) [21]. These cells were maintained in EMEM with EBSS supplemented with 10% FCS, 2 mM L-glutamine, antibiotics and 25 μg/ml Fungizone, 1% sodium pyruvate and 1% MEM vitamin mixture (Cambrex, Haverhill, UK). The subcutaneous haemangioma endothelial cell line, sEnd.1, was derived from a C3H mouse [22]. These cells were maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, antibiotics, 1% sodium pyruvate, 1% MEM vitamin mixture and 1% non-essential amino acids (Invitrogen, Paisley, UK).
Jurkat cells, a transformed human lymphoblastic leukaemia T-cell line [23], were maintained in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine and antibiotics. BALB/c mice primary T cells were purified from spleen cells following incubation with rat anti-mouse class II, anti-CD16/32 and anti-B22 (Caltag, Silverstone, UK) antibodies followed by magnetic separation using sheep antirat beads (Dynal Biotech, Norway). These were maintained overnight in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 5 × 10− 5 M mercaptoethanol (2-ME) and antibiotics.
3.2. Cloning of mouse VCAM-1 gene Whole RNA was extracted from the MCECs using TRIzol® (Life Technologies, UK) according to the manufacturer's instructions. A 5 μg aliquot of total RNA was reverse transcribed into cDNA using oligo dT primer (2.5 mM), 10 mM dNTPs, RNAse inhibitor and reverse transcriptase (GIBCO-BRL, UK). The mouse VCAM-1 gene was PCR-amplified using the primers, 5′-CTCCA CATCTCGAGATGCCTGTGAAGATGGTCG-3′ and 5′-CATTTCAGAATT CAGCTACACTTTGGATTTCTGT-3′ containing Xho1 and EcoR1 restriction sequences at 5′ and 3′ ends, respectively. The gene was cloned into the pcDNA3.1 (−) plasmid (Invitrogen), and the sequence checked. This construct was termed pcDNA3.1/mVCAM-1 (abbreviated in figures to V3).
3.3. Selection and cloning of mouse VCAM-1-specific sequences for vector-based RNA interference Nine target sequences were selected from the published mouse VCAM-1 mRNA sequences (both the coding region and the 3′ non-coding region) (NCBI database accession no. BC029823). Each sequence was 19 nucleotides length (Table 1). The sequences were chosen so that the GC content was 30–50%, and there was no sequence of four or more adenines or thymidines. The sequences were blast-searched against murine EST libraries to ensure that they were not homologous to other sequences. Forward and reverse oligonucleotide primers were designed as described [19] with a nine nucleotide spacer sequence, flanked by BglII and HindIII sequences at the 5′ and 3′ ends respectively. These were annealed and cloned in to the pSUPER vector (Oligoengine, USA) between the BglII and HindIII sites. The negative controls (NC1, NC5, NC19) are specific for Hepatitis C virus mRNA. All constructs were sequenced to confirm correct insertion of the oligonucleotides. For stable transfection, the oligonucleotides encoding si12, si14 and si16 and NC1 were cloned into pSilencer.puro (Ambion Inc., USA). For some experiments an intracellular antibody (E6) consisting of a single chain Fv fused to the KDEL sequence cloned into the pEF/myc/ER plasmid (Invitrogen, USA) was used (manuscript in preparation).
3.4. Transfection Lipofection was carried out using LipofectAmine (Life Technologies, UK) following the manufacturer's protocol optimized for different cell types. For stable transfection the sEnd.1 cell line was transfected with pSilencer.puro vector-based siRNA constructs, selected with Puromycin (4 μg/ml) and cloned by limiting dilution. The sEND.1 cells, and clones derived from them were induced to express surface VCAM-1 molecule by stimulating with 50 ng/ml TNFα for 24 h.
3.5. Flow cytometry Flow cytometric analysis was carried out on cells stained with monoclonal antibodies against mouse VCAM-1, CD40 and PDL1 (all from e-Bioscience, California, USA). FITC-conjugated rabbit anti-rat Ig was used as secondary antibody (Sigma, UK).
3.6. Western blotting Cell lysates were prepared and Western blotted, as described [24]. Blots were probed with monoclonal antibodies against mouse β-actin (Santa Cruz) and VCAM-1.
Sequence name
Starts at
si4 sense si4 antisense si5 sense si5 antisense si8 sense si8 antisense si12 sense si12 antisense si14 sense si14 antisense si15 sense si15 antisense si16 sense si16 antisense si19 sense si19 antisense si23 sense si23 antisense
672 967 1113 1198 1231 1639 2004 2213 2764
Forward primer sequence
Spacer sequence
Reverse primer sequence
5′-GATCCCCGGATCCAGAGATTCAATTCTTCAAGAGAGAATTGAATCTCTGGATCCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGGATCCAGAGATTCAATTCTCTCTTGAAGAATTGAATCTCTGGATCCGGG-3′ 5′-GATCCCCGAACTACAAGTCTACATCTTTCAAGAGAAGATGTAGACTTGTAGTTCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGAACTACAAGTCTACATCTTCTCTTGAAAGATGTAGACTTGTAGTTCGGG-3′ 5′-GATCCCCTGAAGTTCTCCAGCTTCTCTTCAAGAGAGAGAAGCTGGAGAACTTCATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATGAAGTTCTCCAGCTTCTCTCTCTTGAAGAGAAGCTGGAGAACTTCAGGG-3′ 5′-GATCCCCGGAGTTAATCTGATTGGGATTCAAGAGATCCCAATCAGATTAACTCCTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGGAGTTAATCTGATTGGGATCTCTTGAATCCCAATCAGATTAACTCCGGG-3′ 5′-GATCCCCGTGGAATTAGTTGTTCAAGTTCAAGAGACTTGAACAACTAATTCCACTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAAGTGGAATTAGTTGTTCAAGTCTCTTGAACTTGAACAACTAATTCCACGGG-3′ 5′-GATCCCCTTACTGAAGGGGGAGACTATTCAAGAGATAGTCTCCCCCTTCAGTAATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATTACTGAAGGGGGAGACTATCTCTTGAATAGTCTCCCCCTTCAGTAAGGG-3′ 5′-GATCCCCTACTACACTCACCTTCATGTTCAAGAGACATGAAGGTGAGTGTAGTATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATACTACACTCACCTTCATGTCTCTTGAACATGAAGGTGAGTGTAGTAGGG-3′ 5′-GATCCCCCATGGATAATCCTGAAGAATTCAAGAGATTCTTCAGGATTATCCATGTTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAACATGGATAATCCTGAAGAATCTCTTGAATTCTTCAGGATTATCCATGGGG-3′ 5′-GATCCCCTGGTCCCATGATGTGTATGTTCAAGAGACATACACATCATGGGACCATTTTTGGAAA-3′ 5′-AGCTTTTCCAAAAATGGTCCCATGATGTGTATGTCTCTTGAACATACACATCATGGGACCAGGG-3′
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Table 1 siRNA primer sequences selected from mouse VCAM-1 mRNA
The table shows both the sense and antisense oligonucleotides used to make the siRNA constructs. The sequences encoding the forward and reverse sequences for the siRNA are shown, between these is a short sequence making a hairpin loop. The starting nucleotide in the VCAM-1 cDNA sequence is indicated (the VCAM-1 coding region sequences is between 322 and 2541). All of the target sequences are from the coding region except for si23 which has been designed from the 3′ UTR.
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3.7. RT-PCR and real-time PCR Total RNA was extracted using Trizol RNA extraction kit (Invitrogen) from 2– 5 × 106 cells. Total (5 μg) RNA was used for reverse transcription in a final volume of 40 μl. One μl of cDNA was used for semi-quantitative and real-time RT-PCR amplifications. Semi-quantitative RT-PCR amplification was performed for VCAM-1, UPR and ds-RNA responsive genes using the following primers; VCAM-1 sense 5′-ATGACATGCTTGAGCCAGG-3′, antisense 5′-GTGTCTCC TTCTTTGACACT-3′; heavy chain binding protein (BiP) sense 5′-CTGGGTACA TTTGATCTGACTGG-3′, antisense 5′-GCATCCTGGTGGCTTTCCAGCCA TTC-3′; C/EBP homologous protein (CHOP) sense 5′-GTCCAGCTGGGAGCT GGAAG-3′, antisense 5′-CTGGTCAGGCGCTCGATTTCC-3′; growth arrest and DNA damage-inducible protein 45 (GADD45) sense 5′-GAGCAGAAGA CCGAAAGGATG-3′, antisense 5′-CTTCAGTGCAATTTGGTTCAG-3′; Stanniocalcin 2 (STC2) 5′-AGGAGAACGTCGGTGTGATT-3′, antisense 5′CTGTTCACACTGAGCCTGGA-3′; activating transcription factor 4 (ATF4) sense 5′-TCCTGAACAGCGAAGTGTTG-3′, antisense 5′-ACCCATGAGGT TTCAAGTGC-3′; 2 5 oligoadenylate synthetase 1 (OAS1) sense 5′-AGGTGGTA AAGGGTGGCTCC-3′, antisense 5′-ACAACCAGGTCAGCGTCAGAT-3′. Real-time PCR amplification was performed in a lightcycler (Roche) using SYBR green master mix (Sigma-Aldrich Company Limited, Poole, UK) according to the manufacturer's instructions. The PCR protocol included an initial denaturation step (95 °C, 10 min) followed by 40 amplification and quantification cycles (95 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s and a single acquisition at 81 °C), and a melting curve program (65–95 °C). Three sets of primers were used, each set spanning the corresponding sequences selected for that siRNA to give a PCR product of about 200 bp. The primers used were: si12 primers; sense (5′-AGTTCTCCAGCTTCTCTCAGGA-3′), antisense (5′GGGAGATGTCAACAATAAATGG-3′); si14 primers; sense (5′-GCCATCCT CACCTTAATTGCTA-3′), antisense (5′-ATAGCAGCACACGTCAGAA CAA-3′); si16 primers; sense (5′-ATGGGATACCAGCTCCCAAA-3′), antisense (5′-TGCTAATTCCAGCCTCGTTAAT-3′). The relative mRNA expression level was calculated using the standard curve of pcDNA3.1/mVCAM-1.
3.8. Static adhesion assay sEnd.1 cells, either untransfected or transfected with the pSUPER/si plasmid constructs, were seeded in 96-well plates at 1.25 × 104 cells/well and grown to confluence (48 h). Cells were either unstimulated or stimulated with TNFα (50 ng/ml) for 24 h before the assay. Jurkat cells (1–2 × 107) were labelled with Calcein-AM (Molecular probes, UK) at 10 μg/ml for 1 h. After washing with warm PBS, 2.5 × 105 Jurkat cells were added per well for 30 min at 37 °C. Unbound Jurkat cells were gently washed off with three changes of warm PBS, and the fluorescence was measured in a plate reader. Each experiment was performed in quadruplicate.
3.9. Flow adhesion assay The flow chamber adhesion assay was modified from a published method [25]. Briefly, untransfected and stably transfected sEnd.1 clones were grown for 48 h to confluence in 9-cm2 Nunc Slide Flaskettes (Nalge-Nunc International, Roskilde, Denmark), unstimulated or stimulated with TNFα (50 ng/ml) for 24 h before the assay. These were mounted in a parallel plate flow chamber (channel height 0.15 cm). Mouse primary T cells were either unstimulated or stimulated with 10 μg/ml of Con A for 3 h. Jurkat cells or primary T cells were labelled with Calcein-AM as described above, resuspended at 0.25 × 106 cells/ml in RPMI 1640/2% FCS (viscosity 0.007 P) and perfused at 37 °C over the monolayers at a shear stress of 1.5 dyn/cm2 for the Jurkat cells. For the primary T cells, the shear stress used was 1.0 dyn/cm2 for 1 min to allow the cells to cover the monolayer and then 0.75 dyn/cm2 for the rest of the experiment. Experiments were visualized using an inverted Nikon Diaphot 300 fluorescence microscope connected to a colour video camera. Recording commenced after 2 min of perfusion and consisted of a 15 s recording of 10 random fields using a 10× objective; the area of the analysed field was 800 μm × 400 μm. Each experiment was repeated at least three times on separate days.
Analysis was performed using EML Motion Analysis software (Ed Marcus Laboratories, Brighton, MA). The following indices were calculated: the minimum and maximum velocity of individual cells, the number of rolling cells per field and the number of arrested cells per field. Rolling cells were defined as those with a mean velocity of less than 100 μm/s. Arrested cells were defined as those moving less than 5 μm in 10 s.
4. Results 4.1. Isolation of siRNA constructs specific for VCAM-1 Based on the sequence of murine VCAM-1, we designed nine siRNA constructs (Table 1). Candidate siRNA sequences were generated as described in Materials and methods against the entire VCAM-1 sequence, including the untranslated regions, and the nine constructs were chosen randomly from these. Appropriate oligonucleotides were made and successfully cloned into the pSUPER vector [19,26–29]. To determine whether the siRNA constructs inhibited VCAM-1 expression, we co-transfected them into HEK293 cells together with a construct encoding VCAM-1. All 9 constructs inhibited VCAM-1 expression to, or close to, background levels, with no effect of control siRNA constructs (NC5 and NC19) (data not shown). 4.2. Transfection of corneal endothelial cell lines We then transfected the constructs into the MCEC corneal endothelial cell line, which constitutively expresses VCAM-1. The cells were assessed by flow cytometry for the expression of VCAM-1 after 12, 24, 36 and 48 h. Transfection of MCEC cells using the lipofection approach results in around 50% transfection efficiency when assessed with a plasmid encoding EGFP (data not shown). As is shown in Fig. 1A and B for representative constructs at 36 h, and in Fig. 1E for all the constructs, the majority of the siRNA constructs were effective at inhibiting VCAM-1 expression. The degree of inhibition was not complete, as would be expected given the transfection efficiency. Inhibition was seen at all time points, with a trend to maximal inhibition at 48 h. Some constructs (si15 and si19) did not inhibit VCAM-1 expression. Transfection with control constructs (siNC1, siNC5 and siNC19) did not affect VCAM-1 expression. To determine the specificity of VCAM-1 inhibition, the cells were stained for CD40 expression. There were no alterations in CD40 expression with any of the constructs at any of the time points (Fig. 1F). The three constructs that showed greatest inhibition of VCAM-1 expression (si12, si14 and si16) were transfected into MCECs. After 36 h cell lysates were made and analysed by SDS-PAGE and Western blotting for expression of VCAM-1 protein. There was considerable reduction in VCAM-1 protein, with no reduction in cells transfected with a control construct (Fig. 1G). We also transfected the cells with an intracellular antibody that binds mouse VCAM-1 (E6, manuscript in preparation), and showed a reduction in VCAM-1 expression in these cells. The level of VCAM-1 mRNA was determined by real time RT-PCR in MCECs transfected with the constructs. As shown in Fig. 1H (results of one representative primer set, si12) there was downregulation of mRNA levels for most of the constructs, though as with protein expression this was not seen for si15 and si19. Cells transiently transfected with the E6 intracellular anti-VCAM-1 antibody did not show a reduction in VCAM-1 mRNA (not shown), consistent with the post-translational action of this molecule. 4.3. Transfection of vascular endothelial cell lines The vascular endothelial cell line sEnd.1, which is derived from skin haemangioma cells, expresses very low levels of VCAM-1;
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Fig. 1. Reduction of VCAM-1 expression in MCE cells. Flow cytometric analyses of the expression of VCAM-1 or CD40 at 36 h following either no transfection (Untr.) or transfection with negative control, NC1 (A, C) or two representative VCAM-1 specific constructs si12 and si14 (B, D). The shaded profile shows staining of untransfected MCE cells by isotype control antibody. The histograms (E, F) show the mean fluorescence ration (MFR) of intensity of VCAM-1 or CD40 expression 12, 24, 36 and 48 h following transfection with the nine VCAM-1 specific and 3 negative control (NC1, NC5, NC19) siRNA constructs. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three experiments. (G) Lysates of MCEC untransfected, or transfected with NC5, si12, si14 or si16 siRNA constructs and an anti-porcine VCAM-1 scFv construct (E6) that cross-reacts with mouse VCAM-1 were prepared at 36 h post-transfection, separated by SDS-PAGE, western blotted and probed with an anti-mouse VCAM-1 antibody. β-Actin was used as loading control. Densitometric analysis of the VCAM-1 blot shows a siRNA specific reduction (original western blot in inset). Real-time RT-PCR results (H) show mRNA copy-numbers at 36 h following transient transfection with all of the nine specific siRNA constructs and negative controls (NC1, NC5). Results shown are those obtained using one of the representative primer sets, si12. HPRT was used as internal control. P b 0.05 for all VCAM-1 siRNA constructs when compared to MCEC using ANOVA. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
however, following stimulation with TNFα, VCAM-1 expression is upregulated. Cells were transfected with the constructs and the expression of VCAM-1 was determined 24, 36 and 48 h later. When indicated, the cells were activated by treatment with 50 ng/ml TNFα 24 h prior to analysis. There was upregulation of VCAM-1 expression in sEnd.1 cells that were untransfected or transfected with a control siRNA construct and then stimulated with TNFα. This upregulation of VCAM-1 expression was inhibited by all the siRNA constructs, though
to a varying extent (si8, si15 and si19 being less effective than the other constructs) (Fig. 2). We also assessed expression of Programmed Death Ligand 1 (PDL1). This is expressed at low levels on resting sEnd.1 cells, but is upregulated by cytokines. There was increased PDL1 expression (especially at later time points) in all cells transfected with siRNA constructs. This included cells transfected with the control siNC1 construct, indicating that this is an effect of transfection and/or siRNA
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Fig. 2. VCAM-1 and PDL1 expression by cytokine-stimulated sEnd.1 cells following transient transfection with siRNA constructs. (A) The sEnd.1 cells were either untransfected (Untr) or transfected with siRNA constructs (NC1, si12, si14) and then stimulated immediately with TNFα. 36 h later these were analysed for VCAM-1 expression by flow cytometry. The shaded profiles show staining of untransfected cells by isotype control antibody. The sEnd.1 cells were transfected with siRNA constructs and analysed by flow cytometry for VCAM-1 (B) and PDL1 (C) expression at 24, 36 and 48 h. Cells were stimulated with TNFα 24 h prior to analysis. The MFR of unstimulated cells (sEND-Unst) and untransfected cells stimulated with TNFα (sEND-St) is shown. A marked reduction in VCAM-1 expression is seen with all the VCAM-1 specific constructs (P b 0.05 when compared to sEND-St using ANOVA). There is an increase in PDL1 expression for all siRNA constructs (P b 0.05 when compared to sEnd-St using ANOVA). Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
expression, rather than a specific effect of the VCAM-1 siRNA constructs (Fig. 2C). 4.4. Generation of stable vascular cell lines In order to generate stable cell lines expressing siRNA, the constructs were subcloned into pSilencer.puro plasmid. Stable clones of sEND.1 cells containing these constructs were generated and analysed for expression of VCAM-1 following TNFα stimulation. Data from nine representative clones are shown in Fig. 3A (nomenclature: first number indicates the siRNA construct, and the second the clone number). The majority of clones showed little upregulation of VCAM1 expression following stimulation, when compared to either parental sEnd.1 or clones containing control siRNA constructs. Four stable clones (12.2, 12.3, 12.7 and 14.21) were selected for further analysis. The clones expressed normal levels of ICAM-1 (data not shown), and somewhat upregulated PDL1 expression 24 h after TNFα stimulation (Fig. 3B). Stable transfection of VCAM-1 siRNA did not influence PDL1 expression in unstimulated cells. 4.5. T cell adhesion to siRNA expressing cells To study the functional effect of VCAM-1 knockdown, the ability of the human T cell line, Jurkat, to adhere to a monolayer of sEnd.1 cells was determined using a static assay (Fig. 4A). Adherence of Jurkat to parental sEnd.1 cells is dependent on VCAM-1, as shown using an anti-VCAM-1 blocking antibody 429. Blocking ICAM-1 with antibody YN1 also reduced binding of Jurkat cells. The amount of
adhesion of Jurkat cells to 12.2, 12.3 12.7 and 14.21 clones was greatly reduced. While static assays give useful information about the adhesion of cells to monolayers, they are not representative of the situation in vivo, where leukocyte adherence and transmigration occur in the presence of flow. We, therefore, carried out adhesion assays to endothelial monolayers under flow conditions, analysing both the number of cells rolling and those that have undergone arrest. Few Jurkat cells were seen either rolling or firmly adhered to the parental unstimulated sEnd.1 cells (Fig. 4B). However, following stimulation with cytokines there was an increase in the number of arrested cells. This was inhibited by antiVCAM-1 antibody, 429. The adherence of Jurkat cells to 14.21 (following cytokine stimulation) was greatly reduced. There was little rolling of any cells, consistent with the negligible level of E-selectin expression seen 36 h after stimulation of endothelial cells (data not shown). The above experiments were performed in a xenogeneic combination. We, therefore, repeated them using freshly isolated mouse T cells (Fig. 4C). There was some adherence of the murine cells to unstimulated sEnd.1 cells, which was increased by stimulation. However, with the 14.21 clone there was a decrease in adhesion under both stimulated and unstimulated conditions. Taken together these data indicate that inhibition of VCAM-1 expression by siRNA is capable of inhibiting the VCAM-1 dependent adhesion of T cells to endothelial cells. 4.6. Non-specific activation of endothelial cells by siRNA The increase in PDL1 expression following siRNA transfection suggests that the constructs have a non-specific effect on the cells.
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Fig. 3. VCAM-1 expression in stable sEnd.1 clones. The siRNA sequences (NC1, si12, si14 and si16) were cloned into the pSilencer.puro plasmid and the sEnd.1 cells were either untransfected (Untr.) or transfected with these constructs. Following cloning and selection with Puromycin, the cells were analysed for expression of VCAM-1 (A) or PDL1 (B) either without stimulation (Unst.) or 24 h after stimulation with TNFα. Most of the clones show a dramatic reduction of VCAM-1 expression while there was an increase in PDL1 expression (P b 0.05 for all VCAM-1 siRNA clones when compared to Untr. using ANOVA). Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three separate experiments.
Moreover, the sEnd.1 cells stably transfected with the siRNA constructs showed slower growth than untransfected cells (data not shown). In addition, some clones, over time, had a tendency to ‘revert’ with a gradual increase in expression of VCAM-1. These data are consistent with there being a growth disadvantage to cells expressing siRNA. Similar slower growth was also seen with the clones expressing control siRNA. We, therefore, looked for upregulation of stress response molecules. In MCEC cells transiently transfected with the siRNA constructs there was upregulation of BiP, CHOP, GADD45, ATF4 and STC2 as detected by RT-PCR, which was similar whether the cells were transfected with VCAM-1 specific constructs or control constructs (Fig. 5A). However, there was no upregulation of IFNβ following transfection (data not shown). In stable sEND.1 clones there was similar upregulation of the same response elements as well as OAS1 (Fig. 5B).
5. Discussion Interactions between adhesion molecules expressed on endothelium and leukocytes are central to graft rejection, and blocking these interactions can prolong graft survival [6,8,10]. In this study, we sought to determine the effects of reducing VCAM-1 expression using siRNA. We have shown that siRNA can greatly reduce VCAM-1 expression in both corneal endothelial cells
Fig. 4. Static and flow adhesion assay of Jurkat T cells (J6) or mouse primary T cells on sEnd.1 cell monolayers. In all cases except where indicated as unstimulated (Unst.), the endothelial cells and/or the stable clones were stimulated with TNFα 24 h prior to the experiment. (A) sEnd.1 cells were either untransfected (Untr.) or stably transfected with siRNA constructs including the negative control (NC1) grown as monolayers and adherence of Jurkat cells was assessed under static conditions. When indicated, anti-mouse VCAM-1 (429), anti-mouse ICAM1 (YN1) and isotype control antibodies were added. J6 indicates the total fluorescence activity of labelled Jurkat T cells only. Two of the four stable siRNA clones used showed markedly reduced adhesion of Jurkat cells. (B) Jurkat cells were analysed over cytokine activated sEnd.1 endothelial cell monolayers under flow conditions and the number of rolling or arrested cells was determined. There was significantly reduced arrest of Jurkat cells especially on the stable clone 14.21. (C) Primary mouse T cells were also assessed under flow conditions for their rolling and arrest on unstimulated or stimulated sEnd.1 cell monolayers. The stable clone, 14.21 monolayer again showed a statistically significant reduction of arrest of the T cells. Results are expressed as the mean ± S.D. of triplicate cultures and are representative of three independent experiments.
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Fig. 5. Stress responses following siRNA transfection. (A) MCEC were either untransfected or transiently transfected with the siRNA constructs including negative controls (NC1, NC5, NC19). Alternatively (B) sEND.1 cells stably transfected with siRNA constructs were used. RT-PCR for stress markers was performed either 36 h after transfection (MCEC) or 24 h after stimulation with TNFα (sEND-1). Where indicated PC (positive control), cells were treated with Thapsigargin (300 nM) for 10 h. Where indicated ‘No RT’ reverse transcriptase was omitted from MCEC reactions. β-Actin was used as loading control.
(that constitutively express VCAM-1) and vascular endothelial cells (that upregulate VCAM-1 upon activation). This blockade of VCAM-1 expression is effective at reducing T cell adhesion in both static and flow assays. RNA interference has been used to knock down gene expression in different organisms, including plants and Caenorhabditis elegans and Drosophila, and is widely used to study gene function [30,31]. In addition it has been used in preclinical models in a therapeutic context, for example to inhibit the expression of Hepatitis B virus [28,32] and HIV mRNA [26,33]. We would envisage that siRNA could be used to block VCAM-1 expression in the context of transplantation using either gene therapy approaches to express the constructs in endothelial cells [11,29] or in xenotransplantation by making transgenic animals. The use of endothelial-specific promoters [34] could restrict expression to the endothelium. However, for siRNA technology to be successful, it is necessary for its action to be specific. In our study, there is a clear effect of siRNA constructs on cell phenotype, with upregulation of the expression of PDL1 in vascular endothelial cells. In addition, cells containing siRNA constructs (both VCAM-1 specific and control) grew more slowly than cells not containing the constructs (even in the absence of selection Puromycin in the medium), while cells transfected with ‘empty’ vector alone grew normally (data not shown), suggesting some non-specific toxic or inhibitory effect of the siRNA constructs. It is recognised that long dsRNA (N 30 nucleotides) induce a strong interferon response through PKR, OAS1 and TLR3 pathways [18,35–37] inhibiting general protein synthesis. It is often assumed that
synthetic 21 nt siRNA can specifically suppress gene expression in mammalian cells, though it has been reported that transfection with such constructs can lead to an interferon response [38–40]. In order to overcome this problem, plasmid vectors using the pol III H1-RNA or U6 gene promoters have been designed to produce smaller 19 nt siRNA transcripts within the cells [19,41]. The use of the U6 promoter has been reported to nonspecifically induce OAS1 due to the formation of longer double stranded RNA molecules [35,42]. In our study, we used a vector with the pol III H1-RNA promoter. We saw no induction of interferon β response or OAS1 gene induction following transient transfection with the plasmids, although we did observe upregulation of the OAS1 gene in stable clones. However, we did observe a stress response, with increased transcription of CHOP, GADD45 and STC2. These can all be upregulated by increases in ATF4, as observed, which is induced by the production of phosphorylated eukaryotic translation initiation factor (eIF-2α). Phosphorylation of eIF-2α can be induced either by the unfolded protein response (via phosphorylation of PKR-like ER-associated kinase (PERK) [43]) or through activation of phosphorylated protein kinase R (PKR) (induced via the dsRNA pathway) [44]. These stress responses results in inhibition of protein synthesis and RNA cleavage (phosphorylated eIF-2α and OAS1) and apoptosis (CHOP and GADD45). In addition they can increase the transcription of a variety of genes (including BiP). These findings can explain the slower growth of the cells expressing siRNA constructs, as well as the alterations in expression of other proteins. RNA interference has potential for the modulation of cellular phenotype with a view to preventing graft rejection. This includes the inhibition of adhesion molecule expression. However, the non-specific upregulation of stress responses and the consequent non-specific effects on cell phenotype highlight the potential problems with this approach. Acknowledgments This work was funded by the MRC and Action Medical Research. Andrew George was a BBSRC Research Development Fellow. PH Tan was an MRC/Royal College of Surgeon's (Edinburgh) Clinical Research Fellow. References [1] Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:827–72. [2] Jones DA, McIntire LV, Smith CW, Picker LJ. A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions. J Clin Invest 1994;94:2443–50. [3] Briscoe DM, Schoen FJ, Rice GE, Bevilacqua MP, Ganz P, Pober JS. Induced expression of endothelial-leukocyte adhesion molecules in human cardiac allografts. Transplantation 1991;51:537–9. [4] Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokineinduced endothelial protein that binds to lymphocytes. Cell 1989;59: 1203–11. [5] Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol 2003;3 (4):292–303. [6] Stegall MD, Ostrowska A, Haynes J, Karrer F, Kam I, Gill RG. Prolongation of islet allograft survival with an antibody to vascular cell adhesion molecule 1. Surgery Aug 1995;118(2):366–9 [discussion 9-70].
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