SummaryMembrane vesicle (MV) release remains undefined, despite its conservation among replicating Gramnegative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane proteinpeptidoglycan (OM-PG) and OM protein-inner membrane protein (OM-PG-IM) interactions within the envelope structure. Modulation of OM-PG and OM-PG-IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM-PG and OM-PG-IM associations within the envelope structure, thus releasing OM as MVs.

Recently, we identified desmoglein 2 (DSG2) as the main receptor for a group of species B adenoviruses (Ads), including Ad3, a serotype that is widely distributed in the human population. In this study, we have attempted to delineate structural details of Ad3 interaction with DSG2. For CAR- and CD46-interacting Ad serotypes, attachment to cells can be completely blocked by an excess of recombinant fiber knob protein, while soluble Ad3 fiber knob only inefficiently blocks Ad3 infection. We found that the DSG2 interacting domain(s) within Ad3are formed by several fiber knob domains that have to be in the spatial constellation that is present in viral particles. Based on this finding, we generated a small recombinant, self-dimerizing protein containing the Ad3 fiber knob (Ad3-K/S/Kn). Ad3-K/S/Kn bound to DSG2 with high affinity and blocked Ad3 infection. We demonstrated by confocal immunofluorescence and transmission electron microscopy analyses that Ad3-K/S/Kn, through its binding to DSG2, triggered transient opening of intercellular junctions in epithelial cells. Pretreatment of epithelial cells with Ad3-K/S/Kn resulted in increased access to receptors that are localized in or masked by epithelial junctions, e.g. CAR or Her2/neu. Ad3-K/S/Kn treatment released CAR from tight junctions and thus increased transduction of epithelial cells by a serotype Ad5-based vector. Furthermore, pretreatment of Her2/neu-positive breast cancer cells with Ad3-K/S/Kn increased killing of cancer cells by the Her2/neu-targeting monoclonal antibody trastuzumab (Herceptin). This study widens our understading of how Ads achieve high avidity to their receptors and infection of epithelial tissue. The small recombinant protein Ad3-K/S/Kn has practical implications for the therapy of epithelial cancer and gene/drug delivery to normal epithelial tissues.

Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

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Abstract-Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin.Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase-polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin-overexpressing SMCs was significantly reduced by 35Ϯ4% (meanϮSEM, nϭ9; PϽ0.01). Decorin overexpression also induced a higher intimal nuclear density and decreased volume of ECM. Specifically, immunostaining for versican and fibronectin was markedly reduced. In contrast, immunostaining for collagen type I was increased, and electron microscopy confirmed that collagen accumulation was altered. Key Words: proteoglycans Ⅲ smooth muscle cells Ⅲ extracellular matrix Ⅲ gene therapy Ⅲ hyperplasia I ntimal hyperplasia and subsequent arterial stenosis frequently reduce the long-term benefits of therapeutic vascular interventions in patients. The mechanisms leading to the development of neointimal hyperplasia in response to arterial injury involve increased migration, proliferation, and synthesis of the extracellular matrix (ECM) by smooth muscle cells (SMCs). 1,2 Increased migration and proliferation of SMCs, which occur early after injury, are responsible for the formation of a highly cellular intimal lesion. The intimal lesion continues to expand after cell migration and proliferation cease as a result of ECM synthesis and accumulation. The majority of efforts to inhibit intimal hyperplasia have focused on inhibition of SMC proliferation and migration. However, inhibition of ECM accumulation could also be an effective strategy because the ECM constitutes Ϸ80% of the volume of the intimal lesion after 4 weeks. Moreover, the phenotype and behavior of SMCs are influenced through interactions between ECM receptors at the surface of SMCs and specific ECM ligands. [3][4][5][6] Decorin is a member of the family of small leucine-rich proteoglycans. [7][8][9] The core protein of decorin contains 1 dermatan sulfate side chain near the amino terminal of the core protein and 2 independent binding domains for collagen type 1 and transforming growth factor (TGF)-␤ 1 . 10 Decorin was chosen for the present study because it has previously be...