Coxsackie And Adenovirus Receptor Research Articles

A vesicular stomatitis virus glycoprotein epitope-incorporated oncolytic adenovirus overcomes CAR-dependency and shows markedly enhanced cancer cell killing and suppression of tumor growth.

Utility of traditional oncolytic adenovirus (Ad) has been limited due to low expression of coxsackie and adenovirus receptor (CAR) in cancer cells which results in poor infectivity of Ads. Here with an aim of improving the efficiency of Ad's entry to the cell, we generated a novel tropism-expanded oncolytic Ad which contains the epitope of vesicular stomatitis virus glycoprotein (VSVG) at the HI-loop of Ad fiber. We generated 9 variants of oncolytic Ads with varying linkers and partial deletion to the fiber. Only one VSVG epitope-incorporated variant, RdB-1L-VSVG, which contains 1 linker and no deletion to fiber, was produced efficiently. Production of 3-dimensionaly stable fiber in RdB-1L-VSVG was confirmed by immunoblot analysis. RdB-1L-VSVG shows a remarkable improvement in cytotoxicity and total viral yield in cancer cells. RdB-1L-VSVG demonstrates enhanced cytotoxicity in cancer cells with subdued CAR-expression as it can be internalized by an alternate pathway. Competition assays with a CAR-specific antibody (Ab) or VSVG receptor, phosphatidyl serine (PS), reveals that cell internalization of RdB-1L-VSVG is mediated by both CAR and PS. Furthermore, treatment with RdB-1L-VSVG significantly enhanced anti-tumor effect in vivo. These studies demonstrate that the strategy to expand oncolytic Ad tropism may significantly improve therapeutic profile for cancer treatment.

Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response.

The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr.

112. A Novel Oncolytic Adenovirus Based on Human Adenovirus Serotype 17

Most existing oncolytic adenoviruses (AdV) are based on human AdV type 5 from species C (hAdV-C5). Clinical efficacy of hAdV-C5 based oncolytic viruses is limited by variable expression levels of coxsackie- and adenovirus receptor (CAR) in different tumour cells and insufficient replication rates. Additionally, patients exposed to AdV develop serotype specific neutralizing antibodies compromising reapplication. Thus, development of novel oncolytic AdV based on other serotypes may help overcoming these limitations. It may additionally allow repeated dosing in humans and add to the repertoire of antitumour agents in general. We further hypothesised that oncolytic efficacy of the candidate virus can be augmented by expression of RNAi suppressor protein P19 (Lecellier et al., Nature 2005) as has been shown previously for hAdV-C5 (Rauschhuber et al, Sci. Rep. 2013). Based on a computational model AdV of species D including type 17 (hAdV-D17) are predicted to have the highest probability of expressing pre-miRNA compared to other group AdV (Pfeffer et al, Nat. Methods 2005). Moreover, high infection efficiency of EA.hy926 cells, a vascular endothelium tumor cell line that lacks CAR (Jing Liu, unpublished) with AdV17 as well as absence of cross-reactivity with AdVC5 may allow exploitation for AdV-C5-insensitive tumors and systemic therapy. Here we aim at evaluating a novel hAdV-D17-based, p19-containing oncolytic AdV as novel candidate for oncolytic applications in different major tumour cell lines and for toxicity in non-malignant cells. Thus, we cloned hAdV-17 and a P19-containing hAdV-D17 based virus by a novel seamless recombineering technique (Zhang et al, unpublished). In order to allow P19 expression from the adenoviral vector genome, the P19 cDNA was fused via an internal ribosome entry site (IRES) to the late fiber gene. To obtain control viruses, GFP was fused via an IRES to the late fiber gene. After release of the respective recombinant adenoviral genomes from plasmids containing the complete DNA molecule, linearized viral DNA was transfected into A549 cells to perform rescue experiments. To evaluate oncolytic potential we are currently comparing it with wild-type hAdV-D17 and hAdV-C5 using common oncolytic essays. Evaluation in primary tumour cells and eventually in an animal model will allow making assumptions about feasibility of oncolytic virotherapy with this vector. In total we believe that HAdV17-based vectors hold promise for oncolytic applications and may be improved by RNAi-suppression.

154. Targeting Hypoxic and Angiogenic Tumor Microenvironment with pH-Sensitive Oncolytic Adenovirus

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(L-histidine-co-L-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH 6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, VEGF promoter-targeting transcriptional repressor zinc-finger protein-expressing oncolytic Ad (KOX) was used to form complexes. At pH 6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH 7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH sensitive polymer coated Ad complex significantly increase net positive charge upon exposure to hypoxic tumor microenvironment, allowing it to passive target to tumor site. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX.

RGD-modifided oncolytic adenovirus exhibited potent cytotoxic effect on CAR-negative bladder cancer-initiating cells.

Cancer-initiating cell (CIC) is critical in cancer development, maintenance and recurrence. The reverse expression pattern of coxsackie and adenovirus receptor (CAR) and αν integrin in bladder cancer decreases the infection efficiency of adenovirus. We constructed Arg-Gly-Asp (RGD)-modified oncolytic adenovirus, carrying EGFP or TNF-related apoptosis-inducing ligand (TRAIL) gene (OncoAd.RGD-hTERT-EGFP/TRAIL), and applied them to CAR-negative bladder cancer T24 cells and cancer-initiating T24 sphere cells. OncoAd.RGD-hTERT-EGFP had enhanced infection ability and cytotoxic effect on T24 cells and T24 sphere cells, but little cytoxicity on normal urothelial SV-HUC-1 cells compared with the unmodified virus OncoAd.hTERT-EGFP. Notably, OncoAd.RGD-hTERT-TRAIL induced apoptosis in T24 cells and T24 sphere cells. Furthermore, it completely inhibited xenograft initiation established by the oncolytic adenovirus-pretreated T24 sphere cells, and significantly suppressed tumor growth by intratumoral injection. These results provided a promising therapeutic strategy for CAR-negative bladder cancer through targeting CICs.

Short communication: Glucagon-like peptide-2 and coccidiosis alter tight junction gene expression in the gastrointestinal tract of dairy calves

Tight junction (TJ) proteins are integral factors involved in gut barrier function, and therapy with glucagon-like peptide-2 (GLP-2) enhances gut integrity. Our aim was to assess effects of GLP-2 treatment on mRNA expression of 8 TJ complex proteins in the intestine of dairy calves not infected or infected with Eimeria bovis at 11±3d of age. Mucosal epithelium from jejunum, ileum, and cecum was collected at slaughter from Holstein bull calves assigned to 4 groups: noninfected, buffer-treated (n=5); noninfected, GLP-2 treated (n=4); E. bovis-infected, buffer-treated (n=5); and E. bovis-infected, GLP-2-treated (n=4). Infected calves were orally dosed with 100,000 to 200,000 sporulated E. bovis oocysts on d 0; GLP-2-treated calves received 50 µg of GLP-2/kg of body weight subcutaneously twice daily for 10d beginning on d 18; and buffer-treated calves received an equal injection volume of 0.01 M Na bicarbonate buffer. All calves were killed on d 28. The mRNA expression of coxsackie and adenovirus receptor (CXADR), claudins 1, 2, and 4 (CLDN1, CLDN2, and CLDN4), F11 receptor (F11R), junction adhesion molecule 2 (JAM2), occludin (OCLN), and tight junction protein ZO-1 (TJP1) was determined by real-time quantitative PCR. In jejunum and ileum, an interaction of E. bovis infection and GLP-2 treatment on gene expression was noted. In jejunum of noninfected calves, GLP-2 increased CXADR, CLDN2, OCLN, and TJP1 mRNA expression but had no effect on mRNA expression in infected calves. Treatment with GLP-2 also increased tight junction protein ZO-1 protein expression in jejunum of noninfected calves as determined by immunohistochemistry. In ileum, E. bovis decreased expression of JAM2, OCLN, and TJP1 in buffer-treated calves, and GLP-2 increased TJP1 expression in infected calves. In cecum, E. bovis infection reduced expression of CXADR, CLDN4, F11R, and OCLN, and GLP-2 therapy increased expression of CLDN4, F11R, OCLN, and TJP1. Results are consistent with studies in nonruminants showing decreased expression of TJ complex proteins in the intestinal tract during pathogen-induced diarrhea and increased TJ protein expression in intestinal tissues in response to GLP-2 treatment. In conclusion, E. bovis reduces gene expression of TJ proteins primarily in cecum of calves 28d postinfection, and GLP-2 increases expression of selected TJ genes in intestinal tissues. Use of GLP-2 to improve gut barrier function in ruminants during pathogen-induced diarrhea warrants additional study.

PH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX.

Coxsackie and adenovirus receptor is increased in adipose tissue of obese subjects: a role for adenovirus infection?

The coxsackie and adenovirus receptor (CAR) was originally identified as a common receptor for coxsackie B viruses and type C adenoviruses. The objective was to investigate CAR gene expression in human adipose tissue to explore its associations with adipocyte physiology. This was an ex vivo study in 91 visceral adipose tissue (VAT) and 109 sc adipose tissue (SAT) human samples (61 paired) obtained during elective surgical procedures. Patients were recruited at the Endocrinology Service of the Hospital Universitari Dr Josep Trueta. CAR mRNA was measured in human adipose tissue samples and confirmed at the protein level and in adipose tissue fractions. The effects of inflammatory stimuli on CAR gene expression were also evaluated. In paired samples, CAR was 46-fold higher in VAT vs SAT (P < .0001), being higher also at the protein level (P = .04). CAR was predominantly found in stromal vascular cell fractions (SVFs) in both fat depots. CAR mRNA (P = .006) and protein levels (P = .01) in VAT were significantly increased in obese vs nonobese subjects. In fact, CAR mRNA levels in SAT were positively associated with body mass index (r = 0.26; P = .008) and percentage fat mass (r = 0.28; P = .004). In VAT, MGLL, FSP27, AKAP, omentin, TKT, S14, and FABP contributed independently to CAR mRNA variation after adjusting for age and body mass index. Macrophage-conditioned medium led to increased CAR gene expression in mature adipocytes in vitro. The increased expression of CAR in adipose tissue from obese subjects, mainly in SVFs, suggests that CAR might play a role in adipose tissue dysfunction, given its dual associations with adipogenic and inflammatory genes.

Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model.

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

Abstract B50: A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract Oncolytic adenoviruses are able to replicate selectively in cancerous but not normal human cells and are under investigation as potential cancer therapeutics. Unfortunately, mice are poor model systems for therapy with replication competent adenoviruses because murine cells are generally unable to produce infectious viral progeny. In order to model oncolytic adenovirus treatment, researchers have used human tumor xenografts that support viral replication in immunodeficient mice. Because xenografts require an immunodeficient host, xenograft model systems do not reflect a host's adaptive immune response against the virus and cannot model the potential for an immunostimulatory virus to induce an anti-tumor immune response. Using the tumor-selective oncolytic adenovirus TAV-255 previously developed in our laboratory, we find that the murine lung adenocarcinoma cell line LKR-13 supports adenoviral infection and generates infectious viral progeny. Western blotting showed that LKR-13 cells express the coxsackie and adenovirus receptor (CAR) to allow viral infection, and that infected cells express the viral protein E1A. MTT assays, crystal violet staining, and clonogenic assays showed that TAV-255 kills LKR-13 cells in a dose-dependent and time-dependent manner with similar potency and latency of infection as a panel of human cancer cell lines, with more than 50% cell death of murine LKR-13 cells and various human cancer cells (T1, 882 and A549) within 6 days of infection at a multiplicity of infection of 10. It produces infectious progeny from LKR-13 cells at titres similar to other human cancer cell lines. Other tested murine cancer cell lines do not share these features; for example, the CT-26 colon cancer cell line does not express CAR and is resistant TAV-255 infection, while the murine sarcoma cell line F244 is readily infected by adenovirus and produces the viral protein E1A but does not produce infectious progeny. Based on these in-vitro findings, we investigated LKR-13 cells as an in-vivo model system for replicating oncolytic adenoviruses. The LKR-13 cell line is derived from 129/Sv mice, and using fully immunocompetent 129/s4 mice, subcutaneous injection of 5x106 LKR-13 cells led to tumor formation in all injected mice. Intratumoral injection of 3x107 plaque forming units of TAV-255 in established tumors (greater than 200 mm3) caused a significant reduction in tumor growth: Eight days after treatment, tumors in mice treated with TAV-255 were 339 mm3 +/- 26 mm3 (n=3), and tumors in mice treated with PBS were 578 mm3 +/- 49 mm3 (n=5) (p=0.013). This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an anti-tumor immune response. Citation Format: Lingzhi Zhang, Farah Hedjran, Christopher Larson, Min Yan, Tony Reid. A novel immunocompetent murine model for replicating oncolytic adenoviral therapy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B50.

Rethinking Molecular Mimicry in Rheumatic Heart Disease and Autoimmune Myocarditis: Laminin, Collagen IV, CAR, and B1AR as Initial Targets of Disease.

Rationale: Molecular mimicry theory (MMT) suggests that epitope mimicry between pathogens and human proteins can activate autoimmune disease. Group A streptococci (GAS) mimics human cardiac myosin in rheumatic heart disease (RHD) and coxsackie viruses (CX) mimic actin in autoimmune myocarditis (AM). But myosin and actin are immunologically inaccessible and unlikely initial targets. Extracellular cardiac proteins that mimic GAS and CX would be more likely.Objectives: To determine whether extracellular cardiac proteins such as coxsackie and adenovirus receptor (CAR), beta 1 adrenergic receptor (B1AR), CD55/DAF, laminin, and collagen IV mimic GAS, CX, and/or cardiac myosin or actin.Methods: BLAST 2.0 and LALIGN searches of the UniProt protein database were employed to identify potential molecular mimics. Quantitative enzyme-linked immunosorbent assay was used to measure antibody cross-reactivity.Measurements: Similarities were considered to be significant if a sequence contained at least 5 identical amino acids in 10. Antibodies were considered to be cross-reactive if the binding constant had a Kd less than 10-9 M.Main results: Group A streptococci mimics laminin, CAR, and myosin. CX mimics actin and collagen IV and B1AR. The similarity search results are mirrored by antibody cross-reactivities. Additionally, antibodies against laminin recognize antibodies against collagen IV; antibodies against actin recognize antibodies against myosin, and antibodies against GAS recognize antibodies against CX. Thus, there is both mimicry of extracellular proteins and antigenic complementarity between GAS-CX in RHD/AM.Conclusion: Rheumatic heart disease/AM may be due to combined infections of GAS with CX localized at cardiomyocytes that may produce a synergistic, hyperinflammatory response that cross-reacts with laminin, collagen IV, CAR, and/or B1AR. Epitope drift shifts the immune response to myosin and actin after cardiomyocytes become damaged.

Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression

Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and −17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

EP News: Basic and Translational

A heritable component of the Coxsackie and CAR in risk for VF during MI has been established. GWAS for VF during acute MI has led to the identification of a locus on chromosome 21q21 near the CXADR gene. CXADR encodes the coxsackie and adenovirus receptor (CAR), a cell adhesion molecule predominantly located at cardiac IDs. Marsman RF et al (JACC PMID 24291282) investigated the modulatory effect of CAR on ventricular conduction and arrhythmia vulnerability in myocardial ischemia. In human LVs, the risk allele was associated with lower CXADR mRNAs, suggesting that decreased cardiac CAR predisposes to ischemia-induced VF. Hearts from CAR+/- mice displayed ventricular conduction slowing and early onset VTs following LAD ligation. CAR+/- myocytes showed reduced INa specifically at the ID. CAR co-precipitated with NaV1.5, suggesting that CAR affects Na channel function through a physical interaction with NaV1.5. CAR is a novel modifier of ventricular conduction and thus arrhythmia in the MI setting. Genetic determinants such as CAR may constitute future targets for risk stratification of potentially lethal VAs in patients with CAD.

JAM-related proteins in mucosal homeostasis and inflammation

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include junctional adhesion molecules (JAMs) that belong to the cortical thymocyte marker for Xenopus family of proteins. JAM family encompasses three classical members (JAM-A, JAM-B, and JAM-C) and related molecules including JAM4, JAM-like protein, Coxsackie and adenovirus receptor (CAR), CAR-like membrane protein and endothelial cell-selective adhesion molecule. JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration, and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation.

Effect of lovastatin on coxsackievirus B3 infection in human endothelial cells

The coxsackie and adenovirus receptor (CAR) mediates the entry of coxsackievirus B (CVB) and adenovirus into host cells and is, therefore, a key determinant for the molecular pathogenesis of viral diseases such as myocarditis. The aim was to investigate the influence of HMG-CoA reductase inhibitor lovastatin on CAR expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of lovastatin (0.05-5 μmol/l) for up to 48 h. Alterations in CAR expression were examined by quantitative real-time PCR (qRT-PCR) and flow cytometry. In addition, after treatment with 1 μmol/l lovastatin for 48 h, HUVECs were infected for 8 h with CVB3 and virus replication was detected by qRT-PCR using viral-specific TaqMan probes. We found that lovastatin decreases CAR mRNA expression by up to 80% (p < 0.01) and CAR protein expression by up to 19% (p < 0.01), in a concentration-dependent manner. Moreover, virus replication of CVB3 was significantly inhibited after lovastatin treatment (p < 0.05). The signaling mechanism of CAR down-regulation by lovastatin depends on the Rac1/Cdc42 pathway. This study shows for the first time that lovastatin reduces the expression of CAR and subsequently the replication of CVB3 in HUVECs.

Coxsackie and Adenovirus Receptor Is a Modifier of Cardiac Conduction and Arrhythmia Vulnerability in the Setting of Myocardial Ischemia

The aim of this study was to investigate the modulatory effect of the coxsackie and adenovirus receptor (CAR) on ventricular conduction and arrhythmia vulnerability in the setting of myocardial ischemia. A heritable component in the risk of ventricular fibrillation during myocardial infarction has been well established. A recent genome-wide association study of ventricular fibrillation during acute myocardial infarction led to the identification of a locus on chromosome 21q21 (rs2824292) in the vicinity of the CXADR gene. CXADR encodes the CAR, a cell adhesion molecule predominantly located at the intercalated disks of the cardiomyocyte. The correlation between CAR transcript levels and rs2824292 genotype was investigated in human left ventricular samples. Electrophysiological studies and molecular analyses were performed using CAR haploinsufficient (CAR⁺/⁻) mice. In human left ventricular samples, the risk allele at the chr21q21 genome-wide association study locus was associated with lower CXADR messenger ribonucleic acid levels, suggesting that decreased cardiac levels of CAR predispose to ischemia-induced ventricular fibrillation. Hearts from CAR⁺/⁻ mice displayed slowing of ventricular conduction in addition to an earlier onset of ventricular arrhythmias during the early phase of acute myocardial ischemia after ligation of the left anterior descending artery. Expression and distribution of connexin 43 were unaffected, but CAR⁺/⁻ hearts displayed increased arrhythmia susceptibility on pharmacological electrical uncoupling. Patch-clamp analysis of isolated CAR⁺/⁻ myocytes showed reduced sodium current magnitude specifically at the intercalated disk. Moreover, CAR coprecipitated with NaV1.5 in vitro, suggesting that CAR affects sodium channel function through a physical interaction with NaV1.5. CAR is a novel modifier of ventricular conduction and arrhythmia vulnerability in the setting of myocardial ischemia. Genetic determinants of arrhythmia susceptibility (such as CAR) may constitute future targets for risk stratification of potentially lethal ventricular arrhythmias in patients with coronary artery disease.

Aptamer modification improves the adenoviral transduction of malignant glioma cells

Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.

CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis.

Type dependent patterns of human adenovirus persistence in human T‐lymphocyte cell lines

Disseminated adenovirus infections cause significant mortality in stem cell transplanted patients and are suspected to originate from asymptomatic adenovirus persistence ("latency") in lymphocytes. The infection of three human T-lymphocyte lines (Jurkat, PM1, and CEM) with human adenovirus types of species A (HAdV-A31), B (HAdV-B3, -B11), and C (HAdV-C2, -C5) was investigated for 150 days in order to establish in vitro models for adenovirus persistence. HAdV-C5 persisted with continuous production of infectious virus progeny (about 10(7) TCID50 /ml) in PM1 cells. More than 100 copies of HAdV-C5-DNA per cell were detected by real-time PCR but hexon immunostaining showed that only 7.5% of the cells were infected ("carrier state infection"). Coxsackie and adenovirus receptor (CAR) expression was decreased in comparison to mock infected cultures suggesting selection of a semi-permissive subpopulation of PM-1 cells. By contrast, latency of HAdV-DNA (10(-3) -10(-4) copies/cell) without production of infectious virus progeny was observed in HAdV-C2 infection of PM1 and Jurkat, HAdV-A31 infection of PM1, and HAdV-B3 infection of Jurkat cells. In addition, transcription of E1A, DNA polymerase and hexon mRNA was not detected by RT-PCR suggesting an equivalent of clinical "HAdV latency." Persistence of HAdV-DNA was not observed in abortive infections of PM1 cells with HAdV-B3 and -B11 and in productive, lytical infections of Jurkat cells with HAdV-C5, HAdV-B11, and HAdV-A31. In conclusion, lytic and persistent infections with and without production of infectious virus were observed depending on the type of adenovirus. Genetic determinants for viral persistence may be investigated using these newly established infection models.

Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction

Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG.