Extracellular matrix components affect the pattern of protein synthesis of endothelial cells responding to hyperthermia.

The biosynthetic profile of endothelial cells responding to hyperthermia is altered by extracellular matrix components. The extracellular matrix components influence the quantitative expression of members of the HSP70 family and HSP90. The expression of several HSP70 mRNA species, which are strictly stress inducible, are modulated by extracellular matrix components. Both laminin and collagen type IV decrease the amount of HSP70 protein and mRNA expressed by endothelial cells exposed to hyperthermia relative to control cultures attached to virgin plastic. In contrast, both laminin and collagen type IV increased the amount of HSP90 mRNA constitutively expressed by endothelial cells at 37 degrees C. When endothelial cells were exposed to elevated temperatures, these two extracellular matrix proteins decrease the amount of HSP90 mRNA relative to control cultures attached to virgin plastic. Our observations are consistent with the proposal that the extracellular matrix components regulate gene expression and cell behavior in regard to thermotolerance.

Effects of thrombospondin antibody on the recovery of endothelial cells from hyperthermia.

In addition to the increased synthesis of the classical heat-shock proteins (28,000, 71,000, 73,000, 90,000 and 100,000 Mr polypeptides) there is also an increase of thrombospondin in the growth medium of endothelial cells exposed to hyperthermia. The effect of a monoclonal antibody to thrombospondin on the recovery of endothelial cells from hyperthermia as it relates to cytoskeletal organization and cell spreading was assessed. The antibody interacts with the heparin-binding domain of thrombospondin in the extracellular matrix of cells. We report that during recovery from thermal insult at 37 degrees C, intermediate filaments, stress fibres and microtubules show distinct time-recovery characteristics in bovine aortic endothelial cells; that in the presence of this antibody the cytoskeleton is notably altered; that this antibody causes retraction of endothelial cell processes; and that the recovery of the cytoskeleton in endothelial cells exposed to hyperthermia is prevented by the thrombospondin antibody in the time frame examined. Our data suggest that the recovery of cells from heat shock requires the integrity of thrombospondin and its interactions.

Effects of hyperthermia on cell survival and patterns of protein synthesis in endothelial cells from different origins.

Thermotolerance, transient resistance to heat induced by heat itself, is generally thought to be linked to the accumulation of heat-shock proteins in eukaryotic cells. The induction of thermotolerance and the synthesis of heat-shock proteins in primary and passage cultures of bovine aortic endothelium, passage cultures of bovine brain capillaries, and passage cultures of rat epididymal capillaries were examined. Primary and passage cultures of bovine aortic endothelial cells readily acquired thermotolerance; however, passage cultures of rat epididymal capillary cells and bovine brain capillary cells were very heat sensitive. In all endothelial cell types examined except rat epididymal capillary cells, the levels of HSP71, the most inducible of the HSP70 family, correlated well with thermotolerance. With prolonged passage, rat epididymal capillary cells and bovine brain capillary cells lost their ability to acquire heat resistance. Endothelial cells from different origins (aortic endothelium versus capillary endothelium) but from the same species and about the same passage number had a notably different response in terms of thermotolerance and synthesis of proteins after exposure to hyperthermia. The results of this study suggest that, while the expression of HSP71 may be a good indicator of heat resistance, the reverse is not necessarily true. Furthermore, the data show that endothelial cells from different origins are dissimilar in their response to hyperthermia.

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture.

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.

Analysis of 100 kDa polypeptides from coated vesicles of bovine brain: two-dimensional tryptic and chymotryptic maps.

Two-dimensional peptide map analysis was used to determine the structural homology among the '100 kDa'-group of polypeptides. There are at least six distinct polypeptides whose apparent molecular weights are 116, 113, 111, 108, 105 and 100 kDa. The molar ratio of the '100 kDa'-group of polypeptides to three clathrin monomers (equivalent to one triskelion) is 1.2:1. There are three families of polypeptides in the '100 kDa'-group as determined by two-dimensional peptide map analysis. They are 116 and 113 kDa polypeptides, 111, 108, and 105 kDa polypeptides and 100 kDa polypeptide. However, all six polypeptides apparently show a series of homologous peptides. It is suggested that the 100-116 kDa polypeptides may bind to triskelions at the area of homology that is found in the 100-116 kDa polypeptides.

Isolation of bovine aortic endothelial cell plasma membranes: identification of membrane-associated cytoskeletal proteins.

The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane-associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane-associated proteins and extraction with 1% Triton X-100, 60%. At least four heavily glycosylated bands (185-, 165-, 150-, and 130,000 mol-wt) were evident. The Triton-insoluble pellet fraction contained several major polypeptides (30-, 43-, 58-, and 240,000 mol-wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol-st) and vimentin (58,000 mol-wt). Strikingly, vimentin and a 240,000 mol-wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1-like and a 4.1-like protein associated with plasma membranes. The 2.1-like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1-like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti-2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti-protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern. This paper presents several novel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: the presence of two erythroid-like cytoskeletal polypeptides; the presence of vimentin and a 240,000 mol-wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co-elution in a 4:1 mol ratio with a protein perturbant; and the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.

Time-dependent lectin binding to isolated receptors in model membranes.

Binding of lectins to two integral membrane glycoproteins has been measured in lipid bilayer model membranes and in their cell of origin with an eye to clarifying the basis of time-dependence in such processes. Specific binding was monitored as a function of time using an assay that involved membrane exposure to radiolabelled wheat-germ agglutinin or concanavalin A, and subsequent differential centrifugation to remove unbound material. Qualitatively, the time dependence of lectin binding to the isolated receptors in lipid bilayers was found to be similar to that for the same receptors in the intact cell - hence the phenomenon does not depend for its existence upon receptor interaction with other specific native membrane components. Quantitatively, the time-course was sensitive to structural features of the model membrane involved. The results may be understood by viewing the glycopeptide headgroups as deformable structures which rearrange as a direct result of lectin attachment, and it would appear that rearrangement is essential for high-affinity binding. Model membrane structure was examined by light microscopy and freeze-etch electron microscopy in an attempt to assess the applicability of this type of study to a more detailed analysis of the processes involved in lectin binding. Although the freeze-etch technique is a promising one, it was concluded that heterogeneity in receptor arrangement within the lipid bilayer is the most important limitation to correlation of binding curves with membrane structure.

Ganglioside headgroup dynamics.

Gangliosides, spin-labelled specifically on N-acetylneuraminic acid residues or on random-headgroup sugars, have been used to extend previous studies of headgroup behaviour. Headgroup sugar mobility is seen to be homogeneous and relatively unrestricted in a range of systems including three lines of cultured cells. The effects of temperature and pH have been considered. Binding of small quantities of the lectin, wheat germ agglutinin, was found to increase average headgroup mobility for gangliosides in lipid bilayers, most likely as a result of a disordering effect on ganglioside clusters.

Positive cooperativity in a (dissected) lectin-membrane glycoprotein binding event.

A transmembrane glycoprotein, glycophorin, was assembled into large liposomes that may be handled like intact cells. Under appropriate conditions, lectin binding to these simple model cells accurately mimics the positive cooperative behavior commonly reported for binding of various lectins to real cells. Hence we suggest that the simple observation of cooperativity in such a cell-surface recognition event does not necessarily imply an involvement of complex cellular machinery; rather, it may simply reflect interaction of a multivalent ligand with conformationally deformable headgroups.