RESUMEN
Endothelial lesions may lead to the exposure of vascular smooth muscle cells (VSMCs) to the blood flow. In such circumstances VSMCs are exposed to shear stress, an extraordinary mechanical stimulus for this type of cells. Rat VSMCs are cultivated in normal tissue culture plates (statically) or in a cone-plate viscometer (dynamically). Dynamic cultivation leads to a great increase of apoptosis. Immunofluorescence reveals the shear-stress-dependent expression of fas. Apoptosis can be induced by addition of fas ligand-a process which can be blocked by antibodies against either fas or fas ligand. Conditioned medium of dynamically cultivated VSMCs contains fas ligand as the only active apoptosis inducing activity. Apoptosis can be blocked by caspase inhibitors. So the exposure of VSMCs to shear stress leads to apoptosis by the establishment of an autocrine loop of fas and fas ligand-a potential mechanism for the prevention of narrowing of vessel diameter by VSMC proliferation.
Asunto(s)
Apoptosis , Comunicación Autocrina , Glicoproteínas de Membrana/fisiología , Músculo Liso Vascular/metabolismo , Receptor fas/fisiología , Animales , Inhibidores de Caspasas , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Proteína Ligando Fas , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Ratas , Estrés MecánicoRESUMEN
The thrombospondins are a family of extracellular proteins that participate in cell-to-cell and cell-to-matrix communication. They regulate cellular phenotype during tissue genesis and repair. Five family members, each representing a separate gene product, probably exist in most vertebrate species. Like most extracellular proteins, the thrombospondins are composed of several structural domains that are responsible for the numerous biological functions that have been described for this protein family. Considerable progress has been made towards understanding the function of thrombospondins. The role of thrombospondin in the process of apoptosis or programmed cell death has recently come into focus. In this review we will concentrate on the role of thrombospondin-1 in the broad field of apoptotis research.
Asunto(s)
Apoptosis/fisiología , Trombospondina 1/fisiología , Animales , HumanosRESUMEN
Recently it has been shown that vascular endothelial cells (EC) are completely devoid of apoptosis if cultivated under a steady laminar flow and that apoptosis is induced by turning off the flow. An autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex has been identified as the molecular coupling device between flow and apoptosis. Lack of blood flow is a rare and mostly transient phenomenon whereas irregular flow conditions are permanently present at arterial bifurcations and sites of abnormal vessel morphology. Irregular flow conditions are established here either by the action of a cone-and-plate type flow apparatus generating a uniform turbulent flow or in a flow chamber by insertion of a local hindrance creating a zone of unsteady laminar flow with vortex formation and lowered shear stress. In both cases apoptosis is induced either throughout the entire monolayer or restricted to the locally defined area. Flow disturbance and apoptosis are coupled by the described autocrine loop of TSP-1 and the integrin/IAP receptor complex. In vivo atherosclerotic lesions occur predominantly at sites of flow irregularities, which are thought to be pro-atherogenic. Thus we propose a key role of the identified mechanosensitive apoptosis induction for the initiation of atherosclerosis.
Asunto(s)
Apoptosis/fisiología , Endotelio Vascular/citología , Trombospondina 1/fisiología , Antígenos CD , Antígeno CD47 , Proteínas Portadoras , Células Cultivadas , Endotelio Vascular/metabolismo , HumanosRESUMEN
The fas system is present in atherosclerotic lesions. However, its role in the initiation and progression is still unclear. Here we show that in endothelial cells (EC) the expression of the fas receptor is regulated by flow conditions. The EC of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. Recently it was reported that in endothelial cells the lack of hemodynamic forces as well as irregular flow conditions trigger apoptosis by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the alpha(V)beta(3) integrin/integrin-associated protein complex. Here we show that EC cultivated under regular laminar flow conditions are devoid of the fas-receptor whereas cultivation under static conditions as well as under turbulence leads to its expression. Stimulation of the fas-receptor by its ligand increases the amount of apoptotic cells by twofold; the increase can be prevented by blocking the fas-receptor. The availability of the expressed fas receptor for stimulation by its ligand hints at a role as a tool for progression of atherosclerosis.
Asunto(s)
Apoptosis , Endotelio Vascular/metabolismo , Receptor fas/metabolismo , Receptor fas/fisiología , Arteriosclerosis/etiología , Células Cultivadas , Endotelio Vascular/citología , Hemodinámica , Humanos , Estrés MecánicoRESUMEN
Recently, it was reported that the offset of hemodynamic forces induces an unusual pattern of apoptosis in vascular endothelium (1). Although the apoptotic trigger covers all cells and is maintained for a longer time period, only few cells become apoptotic. So, in contrast to common apoptosis inducers, the lack of hemodynamic forces initiates only a low basal level of apoptosis, however steadily increases with time, this way preventing the complete vessel destruction upon an only transient offset of blood flow. The molecular means by which the mechanical stimulus and apoptosis are smoothly coupled have now been identified as an autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex as its receptor. Vascular EC (EC) secrete TSP-1 only in postconfluent static monolayers and not under flow. This also holds true for the IAP whereas the alpha(v)beta(3) integrin is present under static conditions, as well as under flow, assigning the IAP an essential and new switch function in the receptor complex.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis , Proteínas Portadoras/metabolismo , Receptores de Vitronectina/metabolismo , Trombospondina 1/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígeno CD47 , Proteínas Portadoras/inmunología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/metabolismo , Citometría de Flujo , Humanos , Fragmentos de Péptidos/farmacología , Receptores de Vitronectina/inmunología , Estrés MecánicoRESUMEN
Confluent monolayers of primary endothelial cells display a high viability and an apparently constant cell density. However upon prolonged cultivation the monolayer degenerates with increasing numbers of senescent cells finally representing the whole culture. Recently we showed that lack of hemodynamic forces induces apoptosis in organ cultures as well as in confluent monolayers of human umbilical cord vein endothelial cells (HUVEC). The apoptosis started at a low level and was counteracted by a continuous proliferation of the remaining cells. Here we show that the induction of apoptosis by lack of hemodynamic forces is a general characteristic of vascular endothelial cells, valid for endothelial cells from various organs and species: human umbilical cord vein endothelial cells (HUVEC), human microvascular placental endothelial cells (HPEC) and bovine aorta endothelial cells (BAEC). Furthermore apoptosis due to the lack of hemodynamic forces can also be induced in various endothelial cell lines: EA.hy 926 derived from HUVEC and PBMEC-A1 derived from PBMEC. However degeneration of confluent monolayers does not occur with these cell lines even in monolayers kept for several weeks. This indicates that the degeneration of normal endothelial cell monolayers is caused by depletion of the proliferation potential of the endothelial cells.
Asunto(s)
Apoptosis , Endotelio Vascular/fisiología , Hemodinámica/fisiología , Animales , Bromodesoxiuridina/metabolismo , Bovinos , División Celular , Línea Celular Transformada/citología , Línea Celular Transformada/fisiología , Células Cultivadas , Endotelio Vascular/citología , Citometría de Flujo , Colorantes Fluorescentes/química , Humanos , Indoles/química , Placenta/citología , Porcinos , Venas Umbilicales/citologíaRESUMEN
An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 10(2) cells cm(-2) for the adherent cells and 5 × 10(4) cells mL(-1) for the suspension cells. The upper limits are 1-2 × 10(5) cells cm(-2) respectively 8 × 10(6) cells mL(-1).
RESUMEN
The endothelial cells (EC) of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. A shear-stress-dependent modulation has been reported for various endothelial functions, and first attempts were made to characterize the nature of the shear-stress-sensitive receptor. Apoptosis has been induced in cultures of vascular EC upon application of various stimuli characteristic of traumatic or pathogenic events. However, no link between apoptosis and mechanical stimulation has been reported so far-neither for vascular EC nor for other types of cells. Here we show that the lack of hemodynamic forces triggers apoptosis in endothelial cells, hinting at mechanical forces as essential stimuli for the maintenance of blood vessels.