Efficient apical IgG recycling and apical-to-basolateral transcytosis in polarized BeWo cells overexpressing hFcRn.

The human neonatal Fcgamma-receptor (hFcRn) involved in overall maternal-fetal IgG transmission is expressed in placental villous syncytiotrophoblast. However, the role of hFcRn in IgG transport and trafficking across this cell layer is poorly characterized. To gain insight into this mechanism we have overexpressed functional hFcRn in trophoblast-derived BeWo cells (BeWo/hFcRn cells) [Ellinger I, Reischer H, Lehner C, Leitner K, Hunziker W, Fuchs R. Placenta 2005;26:171-82] since parental BeWo cells endogenously express low levels of the receptor. We now demonstrate that hFcRn overexpression differentially affected apical-to-basolateral transcytosis and apical recycling: whereas IgG transcytosis was reduced by 35%, IgG recycling was stimulated by 100% as compared to parental cells, indicating that hFcRn plays a role in both processes. The endosomal compartments involved in hFcRn/IgG transport after apical IgG internalization were then analyzed by confocal immunofluorescence microscopy using compartment specific markers. hFcRn/IgG were found in apical early endosomes, in transferrin recycling compartments and in vesicles near the basolateral plasma membrane, presumably transcytotic vesicles. Neither hFcRn nor IgG was routed to the degradative pathway to lysosomes. These transport and localization data are in accordance with efficient hFcRn-mediated apical IgG recycling and basolateral directed IgG transcytosis in placental trophoblasts.

Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D.

The murine neonatal Fc receptor, FcRn, carries out two functions: materno-fetal IgG delivery and maintenance of serum IgG homeostasis. During human pregnancy maternal IgG is transferred across placental syncytiotrophoblasts presumably by the human homolog of FcRn, hFcRn. Trophoblast-derived BeWo cells express hFcRn endogenously and can be considered as a model system to investigate IgG transport in syncytiotrophoblasts. Using a pulse-chase protocol, we here demonstrate that polarized BeWo cells exhibit not only apical to basolateral transcytosis but also apical IgG recycling. Thus, for the first time we demonstrate that epithelial cells can be involved in both materno-fetal IgG transmission and regulation of serum IgG levels. Lowering the temperature from 37 to 16 degrees C reduced, but did not block, IgG recycling and transcytosis. Microtubule-disruption by nocodazole did not influence transcytosis or apical recycling. Disassembly of filamentous actin by cytochalasin D stimulated apical endocytosis and recycling, while transcytosis remained unaffected. In summary, in BeWo cells apically internalized IgG enters both a transcytotic and recycling pathway. While the transcytotic route is temperature-sensitive but independent from microtubules and actin filaments, the apical recycling pathway is temperature-influenced and stimulated by actin disassembly, suggestive for the involvement of distinct endosome subcompartments in transcytosis and recycling.

Integrins alphavbeta3 and alphavbeta5 mediate VSMC migration and are elevated during neointima formation in the rat aorta.

Neointima formation involves tissue expression of matrix proteins and growth factors. The role of alphavbeta3, but not alphavbeta5 integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of alphavbeta3 and alphavbeta5 integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express alphavbeta3 and alphavbeta5 integrin subunits. The alphavbeta5 integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the alphavbeta3 integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both alphavbeta3 and alphavbeta5 integrins. Selective blocking antibodies for alphavbeta3 and alphavbeta5 inhibited migration of RASMC and HCSMC by more than 60 % (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for alphav, beta3 and beta5 mRNA in uninjured aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of alphav, beta3 and beta5 mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased integrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that alphavbeta3 and alphavbeta5 are elevated during neointima formation in the rat and indicates a novel role for alphavbeta5 participating in mechanisms regulating smooth muscle cell migration.

Expression of CD40 in vascular smooth muscle cells and macrophages is associated with early development of human atherosclerotic lesions.

CD40-CD154-mediated signaling has recently been described as playing a role in cellular functions involved in atherosclerotic processes. CD40 is expressed in macrophages, lymphocytes, endothelial cells, and vascular smooth muscle cells. However, cross-sectional studies investigating the expression of CD40 in atherosclerotic lesions are lacking. In the present study the expression of CD40 was studied in atherosclerotic lesions from 43 patients classified according to the World Health Organization criteria. Serial immunohistologic stainings of human iliac arteries from 43 patients were performed using monoclonal antibodies. Lesions were classified according to World Health Organization criteria, and CD40 expression was analyzed with regard to cell morphology and cellular markers by 2 independent observers. Human atherosclerotic lesions revealed a significant increase in intimal thickness, number of inflammatory infiltrates, and CD40-positive macrophages and vascular smooth muscle cells with progression of the lesions. This increase was most prominent from stage 0 to stage I. A significant correlation between intimal thickness and CD40-positive macrophages (r = 0.75, p <0.0005) and CD40-positive vascular smooth muscle cells (r = 0.81, p <0.0005) was observed. Ligation of the cellular CD40 receptor contributes to inflammatory cellular events in human vascular smooth muscle cells. These data suggest a direct association of CD40 expression in atherosclerotic lesions with early plaque development.

Effects of abciximab and tirofiban on vitronectin receptors in human endothelial and smooth muscle cells.

human umbilical venous endothelial cells. 7E3 binding correlated with alphavbeta3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical venous endothelial cells, these functions were mediated by alphavbeta3 and in human iliac arterial smooth muscle cells by alphavbeta5. In human umbilical venous smooth muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited alphavbeta3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin-cell interactions in vascular cells are mediated via two distinct integrin-receptors, alphavbeta3 and alphavbeta5. Abciximab, which solely inhibits alphavbeta3-mediated cell functions, may be particularly effective in human endothelium and in beta3-integrin expressing vascular smooth muscle cells.

A role for nautilus in the differentiation of muscle precursors.

In the Drosophila embryo, nautilus is expressed in a subset of muscle precursors and differentiated fibers and is capable of inducing muscle-specific transcription, as well as myogenic transformation. In this study, we examine the consequences of nautilus loss-of-function on the development of the somatic musculature. Genetic and molecular characterization of two overlapping deficiencies, Df(3R)nau-9 and Df(3R)nau-11a4, revealed that both of these deficiencies remove the nautilus gene without affecting a common lethal complementation group. Individuals transheterozygous for these deficiencies survive to adulthood, indicating that nautilus is not an essential gene. These embryos are, however, missing a subset of muscle fibers, providing evidence that (1) some muscle loss can be tolerated throughout larval development and (2) nautilus does play a role in muscle development. Examination of muscle precursors in these embryos revealed that nautilus is not required for the formation of muscle precursors, but rather plays a role in their differentiation into mature muscle fibers. Thus, we suggest that nautilus functions in a subset of muscle precursors to implement their specific differentiation programs.