Sean O. Hynes

Simultaneous Expression of Type 1 and Type 2 Lewis Blood Group Antigens by Helicobacter pyloriLipopolysaccharides MOLECULAR MIMICRY BETWEEN H. PYLORILIPOPOLYSACCHARIDES AND HUMAN GASTRIC EPITHELIAL CELL SURFACE GLYCOFORMS

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogenHelicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Leyblood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea(type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pyloriJ223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a singleH. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pyloriadaptation.

Cardiovascular gene therapy: current status and therapeutic potential.

Gene therapy is emerging as a potential treatment option in patients suffering from a wide spectrum of cardiovascular diseases including coronary artery disease, peripheral vascular disease, vein graft failure and in‐stent restenosis. Thus far preclinical studies have shown promise for a wide variety of genes, in particular the delivery of genes encoding growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) to treat ischaemic vascular disease both peripherally and in coronary artery disease. VEGF as well as other genes such as TIMPs have been used to target the development of neointimal hyperplasia to successfully prevent vein graft failure and in‐stent restenosis in animal models. Subsequent phase I trials to examine safety of these therapies have been successful with low levels of serious adverse effects, and albeit in the absence of a placebo group some suggestion of efficacy. Phase 2 studies, which have incorporated a placebo group, have not confirmed this early promise of efficacy. In the next generation of clinical gene therapy trials for cardiovascular disease, many parameters will need to be adjusted in the search for an effective therapy, including the identification of a suitable vector, appropriate gene or genes and an effective vector delivery system for a specific disease target. Here we review the current status of cardiovascular gene therapy and the potential for this approach to become a viable treatment option.

Superparamagnetic iron oxide nanoparticle targeting of MSCs in vascular injury

Vascular occlusion can result in fatal myocardial infarction, stroke or loss of limb in peripheral arterial disease. Interventional balloon angioplasty is a common first line procedure for vascular disease treatment, but long term success is limited by restenosis and neointimal hyperplasia. Cellular therapies have been proposed to mitigate these issues; however efficacy is low, in part due to poor cell retention. We show that magnetic targeting of mesenchymal stem cells gives rise to a 6-fold increase in cell retention following balloon angioplasty in a rabbit model using a clinically applicable permanent magnet. Cells labelled with superparamagnetic iron oxide nanoparticles exhibit no negative effects on cell viability, differentiation or secretion patterns. The increase in stem cell retention leads to a reduction in restenosis three weeks after cell delivery.

Gene-eluting Stents: Adenovirus-mediated Delivery of eNOS to the Blood Vessel Wall Accelerates Re-endothelialization and Inhibits Restenosis

Drug-eluting stents for coronary artery disease results in inhibition of smooth muscle cell (SMC) and endothelial cells which may increase the risk of stent thrombosis. In this study, we attempted to enhance re-endothelialization of deployed stents while simultaneously inhibiting intimal hyperplasia by overexpression of endothelial nitric oxide synthase (eNOS) delivery in the vasculature using an adenovirus gene-eluting stent. Re-endothelialization was significantly greater in vessels obtained from normocholesterolemic animals at day 14 (85.34% +/- 7.38 versus 62.66% +/- 10.49; P < 0.05) and day 28 (91.1% +/- 10 versus 63.1% +/- 22; P < 0.05) and hypercholesterolemic animals (96.97% +/- 3.2 versus 28.33% +/- 38.76; P < 0.05) at day 28 with AdeNOS-eluting stents. At day 28, there was a significant increase in the lumen size [AdeNOS 2.73 mm(2) +/- 1.18, AdbetaGal 0.98 mm(2) +/- 0.98, phosphorylcholine (PC) 1.87 mm(2) +/- 1.18; P < 0.05], and a significant reduction in neointimal formation (AdeNOS 2.32 mm(2) +/- 1.13, AdbetaGal 3.73 mm(2) +/- 0.95, PC 3.2 mm(2) +/- 0.94; P < 0.05), and percent restenosis (AdeNOS 45.23 +/- 20.81, AdbetaGal 79.6 +/- 20.31, PC 70.16 +/- 22.2; P < 0.05) in AdeNOS-stented vessels in comparison with controls from hypercholesterolemic animals, assessed by morphometry and quantitative coronary angiography (AdeNOS 15.95% +/- 7.63, AdbetaGal 56.9% +/- 38.6, PC 58 +/- 34.6; P < 0.05). Stent-based delivery of AdeNOS results in enhanced endothelial regeneration and reduction in neointimal formation as compared with controls. This seems to be a promising treatment strategy for preventing in-stent restenosis (ISR) while simultaneously reducing the risk of stent thrombosis.

Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury.

Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 × 109 PFU of adenoviral(Ad)eNOS, AdiNOS or Adβ-galactosidase (Adβ-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR. The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.07±0.044) and iNOS (0.087±0.086) transduced arteries compared with Adβ-Gal (0.332±0.14) transduced arteries (n=7). In addition, AdeNOS treatment (4.21±3.12% de-endothelialized area) enhanced endothelial regeneration compared to Adβ-Gal treatment (10.05±4.98), while treatment with AdiNOS (25.17±11.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n=7–8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

Background.  The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

Lewis Epitopes on Outer Membrane Vesicles of Relevance to Helicobacter pylori Pathogenesis

Background.  Helicobacter pylori extrudes protein‐ and lipopolysaccharide‐enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host.

Characterisation and differentiation of lactobacilli by lectin typing

Lactobacillus isolates from healthy Estonian and Swedish children were characterised by a lectin typing technique; 56 isolates from six species (L. acidophilus, L. paracasei, L. plantarum, L. fermentum, L. brevis and L. buchneri) were tested. The typing system was based on an agglutination assay with a panel of six commercially available lectins, which were chosen on the basis of their carbohydrate specificities. The isolates were also subjected to proteolytic degradation before lectin typing to decrease auto-agglutination of whole cells in the assay. The 56 isolates were divided into 15 different lectin types by their lectin agglutination patterns. Proteolytic treatment reduced auto-agglutination for the majority of species, apart from L. acidophilus, which remained predominantly auto-agglutinating (eight of nine strains). The system produced stable and reproducible results under standardised culture conditions. Lactobacilli are important bacteria for use as probiotics and this system may supplement current molecular typing techniques and may help in identification of strains that could be useful in this role.

Adenoviral-Mediated Gene Transfer of Nitric Oxide Synthase Isoforms and Vascular Cell Proliferation

Objective: Many vascular diseases are associated with reduced nitric oxide (NO) bioavailability. Nitric oxide synthase (NOS) gene therapy to the vasculature is a possible treatment for vascular disease as a means of increasing NO bioavailability, and this may be achieved using any of the NOS isoforms. The aim of our study was to compare the effects of adenoviral-mediated overexpression of the most commonly used NOS isoforms eNOS and iNOS on vascular cell proliferation. Methods: Human coronary artery smooth muscle cells (HCSMCs) and human umbilical vein endothelial cells (HUVECs) were transduced with adenoviral vectors encoding eNOS or iNOS at a multiplicity of infection of 100. Control cells were exposed to AdNull (empty vector) or diluent alone. Transgene expression was sought by Western blotting. The Greiss assay was used to measure nitrite levels. Cell proliferation was assessed by cell counting on days 0, 3 and 6. Apoptosis was sought using FACS analysis. Angiogenesis was measured using a commercially available in vitro kit. Results: Expression of both isoforms was detected in transduced cells by Western blot at all three time points. NOS transduction resulted in increased nitrite levels with higher levels seen in iNOS- compared to eNOS-transduced cells. Cell proliferation was diminished in AdeNOS- and AdiNOS-transduced cells compared with non-transduced cells on days 3 and 6 in both HCSMCs and HUVECs. Apoptosis was not detected in either cell line with either of the isoforms at any timepoint studied. Both eNOS and iNOS gene transfer caused a reduction in angiogenesis. Conclusions: NOS gene transfer to both endothelial and vascular smooth muscle cells is antiproliferative and antiangiogenic. The biological effect is identical with both isoforms and there is no evidence to support a differential effect on endothelial and vascular smooth muscle cell biology.