Search

AimsEndothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution.Methods and resultsWe carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature.ConclusionThis study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21–32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33–46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states. British Heart Foundation, European Research Council Advanced Grant VASCMIR and British/Israeli Collaborative grant BIRAX.

Intra-arterial administration of an adenovirus serotype 5 (Ad5) vector in a gene therapy trial caused lethal, systemic inflammation in subject 019 with ornithine transcarbamylase deficiency. This unanticipated inflammatory response was absent in another subject receiving the same vector dose and in 16 subjects receiving lower vector doses. We hypothesized that an immune memory to a previous natural adenovirus infection enhanced the immune response to high-dose systemic Ad5 vector, causing the exaggerated immune response in subject 019. To investigate this, we found that rabbit polyclonal sera to Ad5 and pooled human immunoglobulin (Ig) inhibited Ad5 vector transduction of non-immune cells in vitro, but enhanced transduction and activation of human dendritic cells (DCs). Sera from approximately 7% of normal human subjects and 50% of patients treated topically with Ad5 vectors enhanced Ad5 transduction and activation of DCs, apparently from formation of Ig-Ad5 immune complexes and binding to DCs through FcγR. Subject 019's blood substantially increased Ad5-vector activation of human DC primary cultures at levels exceeding those from normal subjects. Although this study is based on one event in a single subject, the results implicate a pre-existing humoral immune response to Ad5 in the lethal systemic inflammatory response that occurred in subject 019.

Background Excessive TGF-β signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their contents and significance have not yet been studied. Here, we aimed to analyse the contents and biological relevance of HPASMC-EVs and their transport to human pulmonary arterial endothelial cells (HPAECs), as well as the potential alteration of these under pathological conditions. Methods We used low-input RNA-Seq to analyse the RNA cargoes sorted into released HPASMC-EVs under basal conditions. We additionally analysed the effects of excessive TGF-β signalling, using TGF-β1 and BMP4, in the transcriptome of HPASMCs and their EVs. We then, for the first time, optimised Cre-loxP technology for its use with primary cells in vitro, directly visualising HPASMC-to-HPAEC communication and protein markers on cells taking up EVs. Furthermore we could analyse alteration of this transport with excessive TGF-β signalling, as well as by other cytokines involved in PAH: IL-1β, TNF-α and VEGFA. Results We were able to detect transcripts from 2417 genes in HPASMC-EVs. Surprisingly, among the 759 enriched in HPASMC-EVs compared to their donor cells, we found Zeb1 and 2 TGF-β superfamily ligands, GDF11 and TGF-β3. Moreover, we identified 90 genes differentially expressed in EVs from cells treated with TGF-β1 compared to EVs in basal conditions, including a subset involved in actin and ECM remodelling, among which were bHLHE40 and palladin. Finally, using Cre-loxP technology we showed cell-to-cell transfer and translation of HPASMC-EV Cre mRNA from HPASMC to HPAECs, effectively evidencing communication via EVs. Furthermore, we found increased number of smooth-muscle actin positive cells on HPAECs that took up HPASMC-EVs. The uptake and translation of mRNA was also higher in activated HPAECs, when stimulated with TGF-β1 or IL-1β. Conclusions HPASMC-EVs are enriched in RNA transcripts that encode genes that could contribute to vascular remodelling and EndoMT during development and PAH, and TGF-β1 up-regulates some that could enhance this effects. These EVs are functionally transported, increasingly taken up by activated HPAECs and contribute to EndoMT, suggesting a potential effect of HPASMC-EVs in TGF-β signalling and other related processes during PAH development.

Long non-coding RNAs (lncRNAs) have structural and functional roles in development and disease. We have previously shown that the LINC00961/SPAAR (small regulatory polypeptide of amino acid response) locus regulates endothelial cell function, and that both the lncRNA and micropeptide counter-regulate angiogenesis. To assess human cardiac cell SPAAR expression, we mined a publicly available scRNSeq dataset and confirmed LINC00961 locus expression and hypoxic response in a murine endothelial cell line. We investigated post-natal growth and development, basal cardiac function, the cardiac functional response, and tissue-specific response to myocardial infarction. To investigate the influence of the LINC00961/SPAAR locus on longitudinal growth, cardiac function, and response to myocardial infarction, we used a novel CRISPR/Cas9 locus knockout mouse line. Data mining suggested that SPAAR is predominantly expressed in human cardiac endothelial cells and fibroblasts, while murine LINC00961 expression is hypoxia-responsive in mouse endothelial cells. LINC00961–/– mice displayed a sex-specific delay in longitudinal growth and development, smaller left ventricular systolic and diastolic areas and volumes, and greater risk area following myocardial infarction compared with wildtype littermates. These data suggest the LINC00961/SPAAR locus contributes to cardiac endothelial cell and fibroblast function and hypoxic response, growth and development, and basal cardiovascular function in adulthood.

Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify the small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1 × 1011 vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting.

Vascular endothelial cells derived from human pluripotent stem cells have substantial potential for the development of novel vascular therapeutics and cell-based therapies for the repair of ischemic damage. To gain maximum benefit from this source of cells, a complete understanding of the changes in gene expression and how they are regulated is required. miRNAs have been demonstrated to play a critical role in controlling stem cell pluripotency and differentiation and are important for mature endothelial cell function. Specific miRNAs that determine stem cell fate have been identified for a number of different cell lineages; however, in the case of differentiation and specification of vascular endothelial cells, this is yet to be fully elucidated.

MicroRNAs (miRNAs) are short noncoding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated miRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES-EC-induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a, and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.

We have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5′ of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

The binding of coagulation factor X (FX) to the hexon of adenovirus (Ad) 5 is pivotal for hepatocyte transduction. However, vectors based on Ad35, a subspecies B Ad, are in development for cancer gene therapy, as Ad35 utilizes CD46 (which is upregulated in many cancers) for transduction. We investigated whether interaction of Ad35 with FX influenced vector tropism using Ad5, Ad35, and Ad5/Ad35 chimeras: Ad5/fiber(f)35, Ad5/penton(p)35/f35, and Ad35/f5. Surface plasmon resonance (SPR) revealed that Ad35 and Ad35/f5 bound FX with approximately tenfold lower affinities than Ad5 hexon–containing viruses, and electron cryomicroscopy (cryo-EM) demonstrated a direct Ad35 hexon:FX interaction. The presence of physiological levels of FX significantly inhibited transduction of vectors containing Ad35 fibers (Ad5/f35, Ad5/p35/f35, and Ad35) in CD46-positive cells. Vectors were intravenously administered to CD46 transgenic mice in the presence and absence of FX-binding protein (X-bp), resulting in reduced liver accumulation for all vectors. Moreover, Ad5/f35 and Ad5/p35/f35 efficiently accumulated in the lung, whereas Ad5 demonstrated poor lung targeting. Additionally, X-bp significantly reduced lung genome accumulation for Ad5/f35 and Ad5/p35/f35, whereas Ad35 was significantly enhanced. In summary, vectors based on the full Ad35 serotype will be useful vectors for selective gene transfer via CD46 due to a weaker FX interaction compared to Ad5.

Recent studies have demonstrated the importance of coagulation factor X (FX) in adenovirus (Ad) serotype 5–mediated liver transduction in vivo. FX binds to the adenovirus hexon hypervariable regions (HVRs). Here, we perform a systematic analysis of FX binding to Ad5 HVRs 5 and 7, identifying domains and amino acids critical for this interaction. We constructed a model of the Ad5-FX interaction using crystallographic and cryo-electron microscopic data to identify contact points. Exchanging Ad5 HVR5 or HVR7 from Ad5 to Ad26 (which does not bind FX) diminished FX binding as analyzed by surface plasmon resonance, gene delivery in vitro, and liver transduction in vivo. Exchanging Ad5-HVR5 for Ad26-HVR5 produced deficient virus maturation. Importantly, defined mutagenesis of just 2 amino acids in Ad5-HVR5 circumvented this and was sufficient to block liver gene transfer. In addition, mutation of 4 amino acids in Ad5-HVR7 or a single mutation at position 451 also blocked FX-mediated effects in vitro and in vivo. We therefore define the regions and amino acids on the Ad5 hexon that bind with high affinity to FX thereby better defining adenovirus infectivity pathways. These vectors may be useful for gene therapy applications where evasion of liver transduction is a prerequisite.

SummaryAdenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive. In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles. Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid. We previously reported that pseudotyping the Ad serotype 5 fiber for that of Ad19p results in reduced hepatic transduction. In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors. It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target. We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection. We engineered the HI loop of Ad19p to accommodate peptide insertions and clones. Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-targeted viruses selectively transducing tubular epithelium and glomeruli, respectively. Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors.

Intravascular delivery of adenovirus (Ad) vectors is being developed for liver-directed gene therapy for targeting disseminated disease in cancer therapeutics and for targeting non-hepatic tissues and organs through vector engineering strategies. The utility of Ad vectors is not limited to serotype 5 (Ad5), and many alternate human serotypes and non-human serotypes of Ad are currently being investigated. Critical to intravascular delivery of Ad is the interaction of the virus with host blood cells and plasma proteins, because immediate contact is observed following injection. Although incompletely understood, recent studies suggest that these interactions are critical in dictating the particle bio-distribution and resulting transduction properties of Ad in vivo. For example, plasma proteins-in particular, vitamin K-dependent coagulation zymogens-are able to directly bind to Ad, and "bridge" the virus to receptors in the liver. Unraveling and characterizing these mechanisms will be of fundamental importance both for understanding basic Ad biology in vivo and for refinement and optimization of Ad vectors for human gene therapy.

The dissection of steps that pattern primitive mesoderm toward the cardiovascular lineages is important to our current understanding of cardiovascular development and can instruct regenerative approaches to heart repair.1 Primitive precursors that can adopt a cardiovascular fate, but also a subset of other mesoderm, are marked by the transcription factor Mesp1 (refs. 2, 3). Later precursors, which selectively give rise to two or more of the cardiovascular cell types (bi- and multipotent cardiovascular progenitor cells), are marked by the transcription factors Nkx2.5 or Isl1, indicators of the first and second heart-forming regions of the embryo (“heart fields”).4,5,6 What has been missing, however, is a molecular feature that uniquely marks cells destined to become cardiomyocytes within the heart. To illustrate, mapping the descendants of cells expressing either Nkx2.5 or Isl1, using the Cre/lox system to indelibly label their progeny in vivo, marks virtually all cell types of the mature heart, including smooth muscle, endothelium, epicardium, and fibroblasts.1 This ubiquity results from expression of Nkx2.5 and Isl1 in the proepicardial organ,7 a specialized structure on the surface of the early heart that gives rise to the external lining of the heart (epicardium) and its derivatives (coronary smooth muscle, coronary endothelial cells, cardiac fibroblasts). In short, what is missing is a marker of the immediate and specific precursor of cardiomyocytes, rather than earlier multipotent cells. A recent report by Jain et al. now shows that an atypical homeodomain transcription factor, Hopx, lacking a motif for direct DNA binding, is restricted to the direct precursors of cardiomyocytes, and not to those of the various other cardiac cells.8

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.

Local adenoviral (Ad)-mediated gene transfer to the carotid artery of the stroke-prone spontaneously hypertensive rat (SHRSP) is successful in improving endothelial function. Here we explored the potential of systemic delivery of Ad encoding endothelial nitric oxide synthase (AdeNOS) to prevent elevation of blood pressure in the SHRSP using both nontargeted and vector targeting approaches. Systemic administration of nontargeted AdeNOS failed to modify the rise in blood pressure in SHRSP when administered during the 12th week of age (n = 5, P = 0.088, F = 3.0), an effect likely to result from sequestration of Ad by the liver. Rerouting Ad transduction using a bispecific antibody (anti-ACE/anti-Ad capsid, Fab9B9) that blocks Ad binding to the coxsackie and adenovirus receptor and simultaneously retargets AdeNOS to the angiotensin-converting enzyme resulted in efficient eNOS overexpression in the lung vasculature and a sustained hypotensive effect (n = 5, P = 0.007, F = 7.9). This study highlights the importance of vector targeting to achieve therapeutic gain and represents the first such study in cardiovascular gene therapy.

Phage display biopanning has been used for a number of applications including ligand generation for targeted drug delivery, targeting gene therapy vectors and identification of protein-protein interaction sites. In this study, a random phage display library was used to isolate peptide ligands to the endothelial protein C receptor (EPCR), identifying 74 different peptide sequences and several motifs. Binding to EPCR was characterized by a solid phase binding assay, demonstrating that 95% of isolated peptides were specific for EPCR. Several homologies with potential relevance to EPCR biology were identified, the most notable being leukolysin (MT-MMP6) and cerastocytin.

Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.1+/-1.2 and 25.5+/-2.2 mm2, respectively) compared to untransfected (6.34+/-0.5 and 20.8+/-1.9 mm2) or lacZ-transfected (6.1+/-0.7 and 19.7+/-1.2 mm2) controls (P0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.

Understanding the molecular and cellular determinants that drive interactions between a host organism and a virus are fundamental questions for both virologists and gene therapists. This rationale is directed from a virology standpoint to gain vital clues into drivers of virus-induced host cell and organ infection and to define potential antiviral strategies. For therapy, such research is utilized to guide strategies for gene-based therapeutics using vectors based on the parental virus’s infectivity profile. In a recent issue of Nature Medicine, Andrew Byrnes and co-workers (Xu et al.1) unveiled a mechanism of adenovirus-mediated evasion against host-derived natural antibodies that is driven through binding of host-derived coagulation factor X (FX) to the adenovirus capsid. This is an important finding that has potentially far-reaching implications for understanding adenovirus-induced pathogenesis and for engineering adenovirus-based therapeutics.

Adenovirus (Ad5) serotype 5 vectors are commonly used for gene transfer. Preclinical studies have shown that their application to systemic gene delivery, however, is limited by their highly efficient uptake in the liver, principally mediated by receptor-binding sites on the fiber shaft and knob domain. Using Ad to target other sites in vivo requires vectors that lack hepatic tropism. We therefore sought to exploit Ad family diversity to isolate vectors that possessed poor hepatic tropism. We pseudotyped the fibers from Ad16 (subgroup B; Ad5/16), Ad19p (subgroup D; Ad5/19p), and Ad37 (subgroup D; Ad5/37) onto Ad5 capsids and assessed infectivity profiles in vitro in multiple cell types and in vivo in rats. In rat, mouse, and human hepatocytes, Ad5/19p and Ad5/37 both possessed a striking lack of hepatic cell infectivity compared with Ad5. Both vectors were, however, able to transduce human vascular endothelial and smooth muscle cells with efficiencies equal to or greater than that of nonmodified Ad5. We evaluated liver uptake in 12-week-old male rats after intravenous injection. In contrast to a vector with the wild-type Ad5 fiber, Ad5, both Ad5/19p and Ad5/37 produced significantly less virion accumulation (measured at 1 hr and 5 days) and transgene expression in the liver. Thus, Ad5/19p and Ad5/37 may be useful platforms for the development of targeted Ad vectors.

Background Matrix metalloproteinases (MMPs) are critical for metastasis of tumor cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural MMP inhibitor, was shown to reduce metastasis in different models. Here, we investigated whether increased TIMP-1 levels in the liver achieved by adenoviral gene transfer will effectively inhibit liver metastasis of two independent tumor cell lines. Method TIMP-1 was transferred with adenoviral vectors into the livers of DBA/2 and Balb/c mice, which were subsequently challenged by hematogenous experimental metastases of the T-cell lymphoma cell line L-CI.5s or the colorectal carcinoma cell line CT-26, respectively. Results MMP-9 expression in the liver was induced upon metastasis in both tumor types. Adenoviral gene transfer led to high transduction efficacy as indicated by lacZ expression in 60% of hepatocytes. TIMP-1, a key inhibitor of MMP-9, was expressed at 105-fold higher levels by adenoviral gene transfer as compared with levels achieved in TIMP-1 transgenic mice, previously shown to be inefficient to reduce T-cell lymphoma metastasis. High local and systemic (serum) levels of TIMP-1 led to substantial (94%) reduction of T-cell lymphoma and colorectal carcinoma (73%) experimental liver metastasis. Conclusions Adenoviral gene transfer led to systemic and local TIMP-1 levels sufficient to inhibit metastasis of a highly aggressive T-cell lymphoma, pointing at the requirement of threshold levels for effective anti-metastatic efficacy. This approach was also efficient in a colon carcinoma solid tumor model. We propose that viral gene transfer of TIMP-1 can provide a suitable defense strategy to prevent metastatic spread to the liver. Copyright © 2004 John Wiley & Sons, Ltd.

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

Patency rates for autologous saphenous vein (SV) conduits used in coronary artery bypass grafts remain poor. Patients with failed grafts are difficult to treat with subsequent interventions, necessitating the development of innovative therapies. Previous studies have suggested that induction of smooth muscle cell (SMC) apoptosis may reduce neointima formation. We overexpressed the proapoptotic gene p53 at the lumenal surface of SV grafts using adenoviral (Ad)-mediated gene transfer in porcine SVs prior to grafting in vivo and analyzed at 7 and 28 days (n = 6 and 7 per group, respectively). p53 overexpression induced a significant upregulation in apoptosis (4 +/- 0.6% for Adp53-infected grafts vs 0.6 +/- 0.1% for Adbeta-gal-infected grafts) and reduced neointimal proliferation by 28 +/- 1% at day 7 postinfection. Adp53-infected grafts had significantly greater lumenal areas than controls at both time points (4.8 +/- 0.6 mm2 vs 2.9 +/- 0.5 mm2 and 10.0 +/- 2.5 mm2 vs 4.2 +/- 1.2 mm2 at 7 and 28 days, respectively). Total graft areas were also increased at 28 days by p53, indicating positive vessel remodeling. Additionally, the thickening of the neointima was significantly reduced by 68 +/- 22% and 28 +/- 3% by p53 overexpression at day 7 and 28, respectively. Importantly, phenotypic changes were maintained at 3 months. Induction of SMC apoptosis by transient p53 overexpression positively influenced vein graft remodeling.

The vascular smooth muscle cell (SMC) is integral to the pathogenesis of neointimal formation associated with late vein graft failure, in-stent restenosis, and transplant arteriopathy. Viral vectors transduce SMC with low efficiency and hence, there is a need for improvement. We aimed to enhance the efficiency and selectivity of gene delivery to human SMC. Targeting ligands were identified using phage display on primary human saphenous vein SMC with linear and cyclic libraries. Two linear peptides, EYHHYNK (EYH) and GETRAPL (GET), were incorporated into the HI loop of adenovirus (Ad) fibers and the capsid protein of adeno-associated virus-2 (AAV-2). Exposure of human venous SMC to EYH-modified (but not the GET-modified) Ad vector resulted in a significant increase in transgene expression levels at short, clinically relevant exposure times. Similarly, the EYH-modified AAV vector resulted in enhanced gene transfer to human venous SMC but not endothelial cells in a time- and dose-dependent manner. The EYH-modified AAV vector also enhanced (up to 70-fold) gene delivery to primary human arterial SMC. Hence, incorporation of EYH into Ad and AAV capsids resulted in a significant and selective enhancement in transduction of SMC and has implications for improving local gene delivery to the vasculature.

1. In rat aortic smooth muscle cells (RASMCs), the putative nuclear factor kappa B (NFkappaB) inhibitor Pyrrolidine dithiocarbamate (PDTC) was found to inhibit lipopolysaccharide (LPS)-stimulated NFkappaB DNA-binding. However, further investigation identified the site of inhibition as being at, or upstream of, the inhibitory kappa B kinases (IKKs) as their kinase activity was substantially reduced. 2. In addition, PDTC potentiated LPS-stimulated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 activity (the downstream target of p38 MAP kinase). 3. Another inhibitor of NFkappaB signalling, the serine protease inhibitor Nalphap-tosyl-L-lysine chloro-methylketone (TLCK), also inhibited LPS-stimulated IKK activity and potentiated JNK activity in response to LPS, suggesting that cross-talk may occur between the NFkappaB and stress-activated protein kinase pathways at the level of IKK or at a common point upstream. 4. Infection of RASMCs with an adenovirus encoding either inhibitory kappa Balpha or a dominant-negative IKKbeta potentiated LPS-stimulated JNK activity. 5. These studies therefore suggest that the loss of NFkappaB DNA-binding and resultant transcriptional activity, rather than the loss of IKK activity, is sufficient to cause an increase in JNK activity. This shows that either pharmacological or molecular inhibition of NFkappaB DNA-binding enhances JNK activation in vascular smooth muscle cells, an effect that may contribute to the pathophysiological effects of LPS.

Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors.

Reorganization of skin during wound healing, inflammatory disorders, or cancer growth is the result of expression changes of multiple genes associated with tissue morphogenesis. We wanted to identify proteins involved in skin remodeling and select those that may be targeted for agonistic or antagonist therapeutic approaches in various disease processes. Full-thickness human skin was grafted to severe combined immunodeficient mice and injected intradermally with 38 different adenoviral vectors inserted with 37 different genes coding for growth factors, cytokines, proteolytic enzymes and their inhibitors, adhesion receptors, oncogenes, and tumor suppressor genes. Responses were characterized for infiltration of inflammatory cells, vascular density, matrix formation, fibroblast-like cell proliferation, and epidermal hyperplasia. Of the 17 growth factor vectors, 16 induced histological changes in human skin. Members of the VEGF and angiopoietin families induced neovascularization. PDGFs and TGF-betas stimulated connective tissue formation, and the chemokines IL-8 and MCP-1 attracted inflammatory neutrophils and monocytes, respectively. The serine protease uPA induced a vascular response similar to that of VEGF. Vectors with adhesion receptors, oncogenes and tumor suppressor genes had, with few exceptions, little effects on skin architecture. The overall results suggest that adenoviral vectors can effectively remodel the architecture of human skin for studies in morphogenesis, inflammatory skin disorders, wound healing, and cancer development.

The last decade has seen substantial advances in the development of gene therapy strategies and vector technology for the treatment of a diverse number of diseases, with a view to translating the successes observed in animal models into the clinic. Perhaps the overwhelming drive for the increase in vascular gene transfer studies is the current lack of successful long-term pharmacological treatments for complex cardiovascular diseases. The increase in cardiovascular disease to epidemic proportions has also led many to conclude that drug therapy may have reached a plateau in its efficacy and that gene therapy may represent a realistic solution to a long-term problem. Here, we discuss gene delivery approaches and target diseases.

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

We have studied the effect of a newly identified tumor suppressor tissue inhibitor of metalloproteinases- 3 (TIMP-3) on the growth of human melanoma and squamous-cell carcinoma (SCC). Adenoviral delivery of the TIMP-3 gene to human melanoma (A2058) and SCC (UT-SCC-7) cells ex vivo inhibited tumorigenesis after subcutaneous (s.c.) injection of the infected cells into SCID/SCID mice. Three daily consecutive intratumoral injections of 1.4×10 9 plaque-forming units (pfu) of TIMP-3 adenovirus (RAdTIMP-3) inhibited the growth of preestablished melanoma and SCC xenografts in SCID/SCID mice, whereas growth of control virus-injected tumors was not affected. The antitumor effect of RAdTIMP-3 was obtained with in vivo adenoviral transduction efficiency of 8–10%, and it was more potent than that of adenovirally delivered p53. Adenovirusmediated expression of TIMP-3 potently reduced gelatinolytic activity, increased the number of apoptotic cells, and inhibited vascularization of melanomas. Escalation of the adenoviral dose to three rounds of three daily consecutive injections with 1.4×10 9 pfu of RAdTIMP-3 every 6 days entirely inhibited growth of injected melanomas for 32 days. Mixing RAdTIMP-3-infected A2058 cells with uninfected cells in 1:1 ratio in culture resulted in death of all cells in 96 hours. Adenovirally delivered TIMP-3 was also expressed by A2058 cells in soluble form into the culture medium, where it exerted a cytotoxic effect on uninfected A2058 cell cultures after relocating to the cell layer. These results identify TIMP-3 as a novel type of secreted tumor suppressor, which has antiinvasive, antiangiogenic, and proapoptotic effects in vivo , and which displays a potent bystander effect validating further exploration of its applicability in human cancer gene therapy.

We investigated the role of polymorphonuclear neutrophil (PMN) proteinases, elastase, and gelatinase B in rat models of acute lung injury. Three groups of rats were studied 6 hours after unilateral instillation of hydrochloric acid (HCl; 0.1 N), lipopolysaccharide (LPS) (4 microg), or saline. The results demonstrated that HCl-induced lung injury, as compared with LPS-induced lung injury, was associated with an increase in permeability (wet/dry weight ratio and proteins in bronchoalveolar lavage fluid). In contrast, there was similar PMN recruitment (in bronchoalveolar lavage fluid and myeloperoxidase activity in lung homogenates) and similar proteinase exocytosis (residual alveolar PMN content of elastase and gelatinase B) in both types of lung injury. In situ zymography, evaluating interstitial protease/inhibitor balance, demonstrated a decrease in gelatinolytic activity in both HCl- and LPS-injured lungs compared with normal lung. The increase in interleukin 6 concentration in lung homogenates, which is observed after both injuries compared with saline-instilled animals, could be involved in up-regulation of tissue inhibitor of matrix metalloproteinase-1, shown by immunocytochemistry to participate in antiproteinase excess. Neither inhibition of alveolar neutrophil influx using a leukocyte elastase inhibitor (EPI-hNE-4) nor inhibition of gelatinase activities by recombinant adenovirus for the human tissue inhibitor of matrix metalloproteinase 1 gene transfer decreased lung edema in HCl-induced injury. These data suggest that PMN proteinases do not contribute to HCl-induced acute lung injury in rats.

Gene transfer may be appropriate for therapeutic protocols targeted at the vascular endothelium. Endothelial dysfunction is the principal phenotype associated with atherosclerosis and hypertension. Oxidative stress has been implicated in the development of endothelial dysfunction. We have explored the ability of overexpressing anti-oxidant genes (superoxide dismutases; SODs) in vitro and in vivo to assess their potential for reversing endothelial dysfunction in a rat model, the stroke-prone spontaneously hypertensive rat (SHRSP). Western blotting and immunofluorescence assays in vitro showed efficient overexpression of MnSOD and ECSOD with respect to localisation to the mitochondria and extracellular surface, respectively. Transgene functional activity was quantified with SOD activity assays. MnSOD and ECSOD overexpression in intact SHRSP vessels in vivo led to endothelial and adventitial overexpression. Pharmacological assessment of transduced vessels following in vivo delivery by basal NO availability quantification demonstrated that the "null" adenovirus and MnSOD adenovirus did not significantly increase NO availability. However, AdECSOD-treated carotid arteries showed a significant increase in NO availability (1.91 +/- 0.04 versus 0.75 +/- 0.08 g/g, n = 6, P = 0.029). In summary, efficient overexpression of ECSOD, but not MnSOD in vivo, results in improved endothelial function in a rat model of hypertension and has important implications for the development of endothelial-based vascular gene therapy.

Gene therapy vectors based on adeno-associated virus-2 (AAV2) offer considerable promise for human gene therapy. Applications for AAV vectors are limited to tissues efficiently transduced by the vector due to its natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. Tropism modification to elevate efficiency and/or selectivity to individual cell types would enhance the scope of AAV for disease therapies. The vascular endothelium is implicitly important in cardiovascular diseases and cancer, but is relatively poorly transduced by AAV vectors. We therefore genetically incorporated the peptide SIGYPLP, which targets endothelial cells (EC), into position I-587 of AAV capsids. SIGYPLP-modified AAV (AAVsig) showed enhanced transduction of human EC compared with AAV with a wild-type capsid (AAVwt), a phenotype independent of heparan sulphate proteoglycan (HSPG) binding. In contrast, AAVsig did not enhance transduction of primary human vascular smooth muscle cells or human hepatocytes, principal targets for AAV vectors in local or systemic gene delivery applications, respectively. Furthermore, infection of EC in the presence of bafilomycin A(2) indicated that intracellular trafficking of AAV particles was altered by targeting AAV by means of SIGYPLP. AAV vectors with enhanced tropism for EC will be useful for diverse gene therapeutics targeted at the vasculature.

MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge Syndrome Critical Region-8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase-3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same “seed sequence” as murine-specific miR-712, and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive “athero-miRs” may provide a new treatment paradigm in atherosclerosis.

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.

Background —Gene transfer to vascular cells is a highly inefficient and nonselective process, defined by the lack of specific cell-surface receptors for both nonviral and viral gene delivery vectors. Methods and Results —We used filamentous phage display to isolate a panel of peptides that have the ability to bind selectively and efficiently to quiescent human umbilical vein endothelial cells (HUVECs) with reduced or negligible binding to nonendothelial cells, including vascular smooth muscle cells and hepatocytes. By direct biopanning on HUVECs and a second approach involving preclearing steps before panning on HUVECs, we isolated and sequenced 140 individual phages and identified 59 peptides. We selected 7 candidates for further investigation by secondary screening of homogeneous phages on a panel of cell types. Using adenovirus-mediated gene transfer as a model gene delivery system, we cloned the peptide SIGYPLP and the positive control peptide KKKKKKK upstream of the S11e single-chain Fv (“adenobody”) directed against the knob domain of the adenovirus to create fusion proteins. Adenovirus-mediated gene transfer via fiber-dependent infection was blocked with S11e, whereas inclusion of the KKKKKKK peptide retargeted gene transfer. The peptide SIGYPLP, however, retargeted gene delivery specifically to endothelial cells with a significantly enhanced efficiency over nontargeted adenovirus and without transduction of nontarget cells. Conclusions —Our study demonstrates the feasibility of using small, novel peptides isolated via phage display to target gene delivery specifically and efficiently to HUVECs and highlights their use for retargeting both viral and nonviral gene transfer to vascular endothelial cells for future clinical applications.

This special issue of Human Gene Therapy is published to coincide with the Annual Meeting of the British Society for Gene and Cell Therapy (BSGCT) taking place in June 2015 at the University of Strathclyde Technology Innovation Centre in Glasgow, United Kingdom. The UK gene and cell therapy community has been at the forefront of international research in the field since its inception, highlighted by the award of 3 of the 12 Human Gene Therapy Pioneer Awards for work in gene and cell therapy to UKbased researchers. Robin Ali of University College London Institute of Ophthalmology was recognized for his highimpact translational research into rare inherited blindness. Adrian Thrasher, past president of BSGCT, of University College London Institute of Child Health is a leader in the field of immunodeficiency disorders, conducting pioneering trials in young patients and deciphering much of the underlying basic science in the development of gene therapy in this arena. Amit Nathwani leads development of clinical gene therapy for hemophilia and has been at the forefront of recognizing the potential application of novel adeno-associated virus serotypes in this arena through translational research. Of course these recent pioneers in the field of gene and cell therapy are the latest contributors to the long and proud scientific tradition of the United Kingdom. In fact, from Charles Darwin’s work in the 1800s, which recognized that species evolved through adaptation via natural selection, to the work of Watson, Crick, and Franklin at the University of Cambridge, which led to the resolution of the double-helix structure of DNA in the 1950s, and latterly the creation of Dolly the Sheep in the 1990s, UK research into genetics and genetic engineering has always been at the forefront of scientific research. Although there is natural synergy between the two research areas, the development of the gene therapy research field generally predates that of stem cell therapy. Gene therapy of course has had its high and low points. At the present time and after much hype in the early days, gene therapy is in the ascendancy again, with clear success in the clinic for a range of serious diseases, significant investment from the pharmaceutical industry, and the relatively recent EU marketing license awarded for the first gene therapy medicine, Glybera. With early recognition of the unique challenges in converting experimental gene therapies into the clinic, the United Kingdom was at the forefront of shaping regulatory policies in this area. The UK government’s Department of Health formed the Gene Therapy Advisory Committee (GTAC), a committee of scientific and medical experts and independent lay members who reviewed submissions for conducting gene therapy clinical trials in the United Kingdom. As well as providing ethical oversight and authorization for clinical trials, they also provided an important and respected scientific advisory function for researchers designing clinical trials to take gene therapies from the lab to the clinic. GTAC has now been dissolved and its functions embedded into the National Research Ethics Service (NRES) of the NHS, highlighting gene therapy’s continuing maturation and acceptance into the mainstream clinical setting in the United Kingdom. Moreover, with the rapid evolution of the cell therapy field, both gene and cell therapies are now classified as advanced medical therapies and are regulated by the UK Medicines and Healthcare Products Regulatory Agency (MHRA), working under EU directives from the Committee for Advanced Therapies at the European Medicines Agency (CAT). Importantly, the advisory aspect first developed for gene therapy through GTAC is still in place for translation of stem cell therapies to the clinic, with the formation of a one-stop shop for advice on regenerative medicines. Therefore, the lessons learned through the history of the development of gene therapy are well placed to support a careful and cautious translation of stem cell therapy from the lab to the clinic. Of course there are still challenges ahead for gene therapy, in particular, bottlenecks in routine availability of GLP and GMP gene therapy vector manufacturing facilities, highlighting that work is still to be done in order to further support and consolidate the field. Therefore, the cell therapy field is well placed to continue learning from the gene therapy field as we move forward.

Human cell culture has become an essential component of biomedical research. The pluripotent phenotype, naturally occurring within the inner cell mass of the early embryo, confers the ability to develop into any cell type of interest. The exploitation of this ability in conjunction with modern genetic engineering tools holds great potential for analysing developmental and adult physiology and pathophysiology as well as for promising an exciting future for regenerative medicine. A broad range of gene delivery systems are available to researchers involved in pluripotent stem cell manipulation. Merits of viral and non-viral systems are driven primarily by the efficiency of stem cell gene transfer. Here, we review the use of viruses (primarily retroviruses, adenoviruses and lentiviruses) and recently optimised non-viral systems for stem cell gene transfer focusing on the recent work on human embryonic stem cells and induced pluripotent stem cells.

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH.

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.

Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmonary arterial smooth muscle cells (bPASMCs) to conditioned media from human PAECs (hPAECs) before and after hypoxic exposure. Serotonin levels were increased in hypoxic PAEC media. Conditioned media evoked bPASMC proliferation, which was greater with hypoxic PAEC media, via a serotonin-dependent mechanism. In vivo, adenoviral vectors targeted to PAECs (utilizing bispecific antibody to angiotensin-converting enzyme (ACE) as the selective targeting system) were used to deliver small hairpin Tph1 RNA sequences in rats. Hypoxic rats developed PAH and increased lung Tph1. PAEC-Tph1 expression and development of PAH were attenuated by our PAEC-Tph1 gene knockdown strategy. These results demonstrate that hypoxia induces Tph1 activity and selective knockdown of PAEC-Tph1 attenuates hypoxia-induced PAH in rats. Further investigation of pulmonary endothelial-specific Tph1 inhibition via gene interventions is warranted.

Autologous saphenous vein is commonly used as a conduit to bypass atherosclerotic lesions in coronary and femoral arteries. Despite the wide use of arterial conduits, which are less susceptible to complications and failure, as alternative conduits, the saphenous vein will continue to be used in coronary artery bypass grafting until acceptable alternative approaches are evaluated. Hence, preservation of vein graft patency is essential for the long-term success. Gene therapy is attractive in this setting as an ex-vivo technology to genetically manipulate the conduit before grafting. The use of safe and efficient vectors for delivery is a necessity as well as a strategy to improve patency in the long term. Here, we review the current clinical practice, the pathogenesis of bypass graft failure and adenovirus-mediated gene therapy strategies designed to improve late vein graft failure by modulation of smooth muscle cells in the vein wall.

The development of adenoviral vectors for intravascular (i.v.) delivery will require improvements to their in vivo safety and efficacy. The hypervariable regions (HVRs) of the Ad5 hexon are a target for neutralizing antibodies, but also interact with factor X (FX), facilitating hepatocyte transduction. Ad48, a species D adenovirus, does not bind FX and has low seroprevalence. Therefore, it has been suggested that Ad5HVR48(1-7), a hexon-chimeric vector featuring the seven HVRs from Ad48, should display advantageous properties for gene therapy, by evading pre-existing Ad5 immunity and blocking FX interactions. We investigated the in vivo biodistribution of Ad5, Ad5HVR48(1-7), and Ad48 following i.v. delivery. Ad5HVR48(1-7) displayed reduced hepatocyte transduction and accumulation in Kupffer cells (KCs), but triggered a robust proinflammatory response, even at relatively low doses of vector. We detected elevated serum transaminases (48 hours) and increased numbers of periportal CD11b(+)/Gr-1(+) cells in the livers of Ad5HVR48(1-7)-treated animals following i.v., but not intramuscular (i.m.), delivery. In contrast, Ad48 did not elevate transaminases or result in the accumulation of CD11b(+)/Gr-1(+) cells. Collectively, these findings suggest that substantial hexon modifications can lead to unexpected properties which cannot be predicted from parental viruses. Therefore, refined mutations may be preferential for the successful development of targeted vector systems which require i.v. administration.