William H. J. Douglas

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

SummaryAngiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells.

Effects of d-valine on pulmonary artery endothelial cell morphology and function in cell culture

The effects of D-valine on the cell culture of bovine pulmonary artery endothelial cells were studied using D-valine-modified Minimal Essential Medium (MEM). D-Valine-treated cultures (46-920 mg/l) were compared with replicate cells grown in L-valine (46 mg/l)-MEM. All media were supplemented with 15% fetal bovine serum (FBS). Endothelial cells were grown for 14 passages with split ratios varying from 1:3 to 1:6. Unlike cells grown in L-valine MEM, cells grown in D-valine MEM did not become contaminated by the growth fibroblasts in primary cultures. D-Valine-treated cells were found to grow in cobblestone array, exhibit contact inhibition and strongly express factor-VIII antigen (F-VIII). D-Valine-grown cells produced PGI2 in greater proportion to PGE2, both constitutively and when stimulated by bradykinin, on comparison with cells grown in L-valine. In addition, cells grown in L-valine, although able to express factor VIII, were not comparable to D-valine cells with respect to other parameters assayed (morphology and growth as a monolayer).

Visualization of cellular aggregates cultured on a three dimensional collagen sponge matrix.

SummaryA one-step vital stain is described for the macroscopic visualization of histotypic cell aggregates in fetal rat lung organotypic cultures. Organotypic cultures are incubated in 0.05-0.1% 2,3,5′-triphenyl tetrazolium chloride (TTC) in culture medium (37°C). Living cells reduce the tetrazole to a water-insoluble red colored formazan. Cell aggregates appear as densely stained foci against the lighter background of the Gelfoam substrate. Stained cultures may be scanned macroscopically to determine the degree of reaggregation and assess cell viability. Identification of aggregates by TTC staining improves the efficiency of tissue processing for electron microscopy and does not alter the ultrastructural appearance of the cultured cells.

[10] Monolayer culture techniques

Publisher Summary This chapter discusses the monolayer cell culture techniques, which are frequently established from single cell suspensions prepared by the enzymic dissociation of organ fragments. The cell preparation is inoculated into a culture vessel containing fluid medium and incubated in a controlled atmosphere. The resultant primary culture is a mixed cell population, which contains many of the cell types present in the tissue of origin. Cell separation and isolation methods such as density gradient centrifugation, electrophoresis, or affinity column separation can be applied to the initial cell suspension prior to culture. Fibroblasts replicate faster than most cell types. Dilution plating at clonal density five is used to separate cell types so that colonies which develop may be physically isolated and then subcultured as pure populations. Basic monolayer cell culture techniques are well established and accessible.

Effect of dexamethasone upon surfactant phosphatidylcholine and phosphatidylglycerol synthesis in organotypic cultures of type II cells

Organotypic cultures of pulmonary type II epithelial cells were treated with dexamethasone at concentrations between 10(-10) and 10(-5) M for 48 h followed by a 3 h incubation in 5.6 mM [U-14C]glucose. A surfactant and a residual fraction was isolated from the cultures by discontinuous sucrose gradient centrifugation. Phosphatidylcholine and phosphatidylglycerol were purified from each fraction and analyzed for total content. The specific activity of each phospholipid was measured as an index of the rate of synthesis. Dexamethasone treatment produced a dose-dependent increase in synthesis and content of surfactant phosphatidylcholine, with a maximum response occurring at 10(-6) M dexamethasone. At concentrations of 10(-5) M, dexamethasone ceased to produce a significant stimulation. Dexamethasone produced an increase in surfactant phosphatidylglycerol synthesis only at a concentration of 10(-8) M and higher. There was not a significant effect upon the content or rate of synthesis of phosphatidylcholine or phosphatidylglycerol in the residual fraction at any of the dexamethasone concentrations tested.

Ultrastructural changes of bovine pulmonary artery endothelial cells irradiated in vitro with a 137Cs source

Bovine pulmonary artery endothelial cells in culture were evaluated by phase-contrast and electron microscopy at various times after being irradiated with 137Cs in vitro. Cells irradiated prior to reaching confluency showed vacuolization aand increased numbers of lysosomes beginning at 48 hr after irradiation with 300-500 rad and at 24 hr after irradiation with 1500-5000 rad. After 7 days the morphological changes appeared to be reversible for cells receiving the lower doses, but were progressive for higher doses of radiation. The same qualitative changes, with a delayed onset, were observed for cells irradiated at confluency. An observed decrease in the endoplasmic reticulum and polysomes occurred only late in the course of radiation injury. There was no observable structural alteration of mitochondria even when there was evidence of otherwise marked cytoplasmic injury. We conclude that structural changes of the lysosomes constitute an early phase of injury by irradiation of the endothelial cell in culture, while decreases in endoplasmic reticulum and polysomes occur relatively late. The mitochondrial structure of the endothelial cell appears to be relatively resistant to radiation. All morphological changes occur subsequent to impaired transport of alpha-aminoisobutyric acid, which is observed within 6 hr as previously reported.

Ultrastructural changes in bovine pulmonary artery endothelial cells exposed to 80% O2 in vitro.

SummaryBovine pulmonary artery endothelial cells in culture were exposed for up to 7 d to a gas mixture containing 80% O2, 5% CO2, and 15% N2 (hyperoxia) and were compared by phase contrast and electron microscopy to cells exposed to a gas mixture containing 20% O2, 5% CO2, and 75% N2. Cells exposed to hyperoxia became enlarged and showed vacuolization and increased lysosomes within 24 to 48 h. These changes were progressive over the 7 d period of exposure. Between 3 and 7 d of exposure to hyperoxia the cells showed reductions in polysomes and endoplasmic reticulum. Despite the other marked cytoplasmic changes, the appearance of mitochondria of oxygen-exposed cells remained unchanged from those of air-exposed cells throughout the 7 d period. Preconfluent and confluent cells responded qualitatively similarly to hyperoxia, but morphological evidence of injury occurred more rapidly for preconfluent cells. We conclude that the initial early structural injury of the endothelial cell exposed to hyperoxia occurs in lysosomes and that the mitochondrial structure is relatively resistant to injury.

[1] Physical aspects of a tissue culture laboratory

Publisher Summary This chapter discusses the physical aspects of a cell and tissue culture laboratory, which includes (1) cleaning and sterilization facilities, (2) media preparation and storage facilities, (3) work area for aseptic manipulation of cell cultures, and (4) equipment for routine cell maintenance. The major component of media and other reagents for the propagation of cells in culture is water. Basic tissue culture operations require an incubator, microscope, cell repository materials, and culture vessels to grow the cells. The artificial capillaries are semipermeable cellulose acetate or polycarbonate membranes through which tissue culture media is constantly perfused. Few specific recommendations are given in the chapter because the absolute requirements for any individual program depends on the existing facilities, the projected scope of the program and the type of cells to be cultured and their intended use. Cleanliness of glassware, purity of water and other reagents, and maintenance of sterility will ultimately determine the reliability of any cell culture system. The information presented is a guide to what is necessary to achieve these basic goals.

Chapter 5 Maintenance of Human and Rat Pulmonary Type II Cells in an Organotypic Culture System

Publisher Summary This chapter discusses the maintenance of human and rat pulmonary type II cells in an organotypic culture system. The lungs of mammals and some inframammalian vertebrates contain a potent surface-active material that coats the alveolar surface, decreases the surface tension, and thus stabilizes the alveoli against collapse. The presence of this pulmonary surfactant is essential for normal pulmonary function, and its absence or diminution is an important etiological factor in respiratory distress syndrome. The mammalian pulmonary alveolus is lined with an endodermally derived epithelium consisting of two cell types: type I and type II pulmonary epithelial cells. The human fetal lung at 18–20 weeks of gestation contains epithelial tubules instead of pulmonary alveoli. The tubules are comprised of glycogen-rich epithelial cells containing very few lamellar bodies. The phospholipid composition of surfactant isolated from organotypic cultures is compared to that isolated from adult rat lung. Fatty acid analysis of surfactant phosphatidylcholine from organotypic cultures demonstrates that myristate, palmitate, and stearate are the major fatty acids present.