Endothelins produced by endothelial cells promote collagen gel contraction by fibroblasts

Endothelial 1 (E1) is identified as an endothelial cell secreted factor that stimulates collagen gel contraction by fibroblasts. This identification is based on (a) co-localization of stimulatory activity in endothelial cell conditioned media with synthetic E1 in reversed phase analysis; (b) removal of the activity from conditioned media with antiserum directed against E1; and (c) the activity of synthetic E1. Treatment of endothelial cell conditioned media with immobilized anti-E1 antibodies removed 59% of the activity from the pool suggesting that E1 is the major contraction promoter in endothelial cell conditioned medium. The mechanism of action of E1 is shown to be different from serum in that E1-promoted contraction is dependent upon the synthesis of an unknown effector protein. Synthetic E1 is shown to be a potent promoter of gel contraction with half-maximal activity occurring at 32 pM. Two other endothelins, E2 and VIC, are slightly less active than E1. A fourth endothelin species, E3, is substantially less active. A comparison of E1 with other contraction promoting peptides revealed that E1 and platelet-derived growth factor are essentially equal in specific activity, whereas TGF beta is approximately 50-fold more potent.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Adhesion of lymphoid cells to fibroblasts in tissue culture

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.     

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions

Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with⁵¹Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H₂O₂ became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805.     

Method for the quantitative study of the growth of fibroblastic cells in tissue culture

The method is elaborated for a quantitative evaluation of hypodermic fibroblast element growth in tissue culture of albino rats. The method is based on determination of the growth activity and intensity for each of the explant growth zones (compact, necwork-like zones and zone of migrating elements). The method permits a rapid and an exact evaluation of the influence of different substances added to the culture medium.     

Formation of a barrier by brain microvessel endothelial cells in culture

Endothelial cells (EC) isolated from bovine brain microvessels produce a continuous monolayer when grown in primary culture. The EC are joined together by tight junctions and contain few pinocytotic vesicles. Horseradish peroxidase (HRP) is unable to penetrate this in vitro barrier system. Exposure of the cells to 1.6 M arabinose produces a reversible separation of the tight junctions with penetration of HRP across the monolayer in a pattern similar to that observed in animals after infusion of hyperosmotic solutions into the carotid artery. The behavior of brain microvascular cells in culture suggest that they retain properties important to the formation of the blood-brain barrier.     

Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells

We have compared the production of prostaglandins in fibroblast-like cells and endothelial cells in culture. Of the fibroblasts studied $\text{10T}\text{1}\text{2}$, SHE, BP6T and KD produce significant amounts of PGI₂, PGE₂ and PGF_2F2 under optimal culture conditions, but only 3T3 and BHK produce TxA₂ in addition to PGI₂. The adult bovine aortic endothelial cells (ABAE) and fetal bovine heart endothelium (FBHE) synthesise PGI₂ but not TxA₂, either from endogenous or exogenous substrates. Both cultured endothelial cells and fibroblasts apparently lack 15-hydroxyprostaglandin dehydrogenase pathway and the ability to convert 6-Keto PGF_1α into 6-Keto PGE₁. PGI₂ production by ABAE was 3–5 times that of FBHE, about twice that of SHE cells and 6–8 times that of $\text{10T}\text{1}\text{2}$ or BP6T cells. Supernatants or media obtained from these cells inhibited aggregation of human platelet-rich plasma, a known biological effect of PGI₂. This effect was abolished when cell monolayers were preincubated with indomethacin or tranylcypromine. RIA and chromatographic data of 6-Keto PGF_1α from these experiments confirmed that the inhibition of platelet aggregation was due to the formation of PGI₂. The production of all prostanoids by endothelial cells or fibroflasts was significantly higher during the exponential phase of growth as compared to confluent monolayers. We propose that fibroblasts $\text{10T}\text{1}\text{2}$ or SHE can serve as useful experimental models for the study of metabolism and transport of PGI₂ and/or TxA₂ in cells of nonendothelial nature.     

A study of mitochondrial-extramitochondrial interactions in giant tissue culture cells by microfluorimetry-microelectrophoresis

Histochemistry and Cell Biology -     

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.     

Motility of mouse fibroblasts in tissue culture.

The growth and motion of mouse L-cells in vitro have been studied by means of time-lapse photography. In particular, the mitotic period and the motility, defined in terms of [R2], the mean square displacement of an ensemble of cells, have been measured as a function of temperature. The motility is a function of the phase of the cell cycle. For approximately the first one-eighth of the mitotic period the motility is well described as a random walk with persistence, the duration of the persistence being determined by the time of extension of the filopodic spindle. The temperature dependence of the diffusion constant follows the Arrhenius factor. The mitotic period, which varies exponentially as (1/T), exhibits a large variance, and the time difference in replication of daughter pairs follows approximately a Poisson distribution with a mean difference of 138 min at T = 37 degrees C. There is no evidence of mirror symmetry in the motion of daughter pairs for fibroblast cells plated in vitro in Corning tissue culture flasks.     

Proteomic study of human umbilical vein endothelial cells in culture

The endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (~ 30 nug), the association of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, liquid chromatography-tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F-actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione-S-transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2-D maps of HUVECs will be soon available on the web at http://www. huvec.com.     

Differences in proteins secreted by human fibroblasts and muscle cells in culture

Cell cultures of human skin fibroblasts, myoblasts, and fused muscle cells were grown in the presence of [¹⁴C]leucine or a mixture of [¹⁴C]amino acids. The proteins synthesised and secreted or leaked into the culture medium during radio-labelling were separated by one and two-dimensional PAGE and detected by fluorography. Four major bands of M_r 54 kD, 52 kD, 51 kD, and 49 kD were present at greatly increased concentration in fibroblast media. These fibroblast-specific polypeptides can be readily detected in myoblast/fibroblast cocultures with fibroblast content as low as 5%.