Phosphatidyl choline and the growth in serum-free medium of vascular endothelial and smooth muscle cells,and corneal endothelial cells

Liposomes made by sonication of egg yolk phosphatidyl choline support the proliferation of low-density bovine vascular and corneal endothelial cells, and vascular smooth muscle cells maintained on basement laminacoated dishes and exposed to a defined medium supplemented with transferrin. The optimal growth-promoting effect of phosphatidyl choline was observed at concentrations of 25 μg/ml for low-density cultures of vascular smooth muscle cells, and 100 μg/ml for vascular and corneal endothelial cells. The growth rate and final cell density of vascular endothelial cells exposed to a synthetic medium supplemented with transferrin and either high-density lipoproteins or phosphatidyl choline has been compared. Although cultures exposed to phosphatidyl choline reached a final cell density similar to that of cultures exposed to high-density lipoproteins, they had a longer average doubling time (17 h vs. 12 h) during their logarithmic growth phase and a shorter lifespan (17 generations vs. 30 generations). Similar observations were made in the case of vascular smooth muscle cells or bovine corneal endothelial cells maintained in medium supplemented with transferrin, fibroblast growth factor (FGF) or epidermal growth factor (EGF), and insulin and exposed to either high-density lipoproteins or phosphatidyl choline. Since phosphatidyl choline can, for the most part, replace highdensity lipoproteins in supporting the proliferation of various cell types, it is likely that the growth stimulating signal conveyed by high-density lipoproteins is associated with its polar lipid fraction, which is composed mostly of phosphatidyl cholines.     

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 microgram/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum. Early passage (P1-P3) bovine adrenal cortex cells cultured in serum-free medium responded to ACTH (10(-8)M) with increased 11-deoxycortisol production; this effect was not observed in later passage cells (P7-P15). The cells' ability to utilize LDL-derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum-free medium. HDL, although also able to increase steroid production in early-passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents. The life span of bovine adrenal cortex cells grown in the serum-free medium on fibronectin (FN)- versus ECM-coated dishes was compared. Cells seeded in serum-containing medium and grown in serum-free medium had a life span of 34 versus 60 generations when maintained on fibronectin- or ECM-coated dishes, respectively. Cells seeded in the complete absence of serum in the serum-free medium on ECM- or fibronectin-coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin-coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.     

Long-term preservation of chilled canine semen: effect of commercial and laboratory prepared extenders

The present study was conducted to evaluate chilled semen conservation over time in 3 commercial and 4 laboratory prepared extenders, including a new Tris-glucose extender. The beneficial effect of adding egg yolk to these media was also analyzed. The effects of these extenders on motility and acrosome reaction were characterized objectively using a computer-aided semen analyzer and the chlortetracycline staining, respectively. No significant differences were observed when comparing the different commercial extenders without egg yolk, but addition of egg yolk improved all motility parameters significantly (preservation of 50% of motility was observed at 3.2+/-1, 2.9+/-0.5, 2.3+/-0.5, 8.5+/-0.2, 5.4+/-1.1, 5.2+/-0.4 d, for Biladyl, green extender and fresh-phos extenders without and with egg yolk, respectively). Motility parameters were best preserved in egg yolk supplemented Biladyl extender with a mean percentage of 86.3+/-10.5 motile spermatozoa after 7 d at 4 degrees C. Efficacy of egg yolk-supplemented commercial extenders on sperm motility at 4 degrees C was (in decreasing order) as follows: Biladyl > green extender > fresh-phos. However, high quality motility and the percentage of motile spermatozoa were highest with some of the laboratory prepared extenders: a 50% conservation rate of motile spermatozoa was observed following the use of supplemented egg yolk extenders. These are classified in decreasing order as follows: Tris-glucose (13+/-1 d) > Tris-fructose (9.7+/-0.6) > EDTA (4.+/-0.6 d) > Tris-bes (3.6+/-1.1 d). A low concentration of motile spermatozoa was still observed in the Tris-glucose egg yolk extender 16 d after collection, clearly demonstrating the importance of the medium and the beneficial effect of egg yolk on sperm motility of 4 degrees C chilled semen. Similar effects of extender were observed for acrosome reactions. Egg yolk clearly had a protective effect reducing acrosome reactions significantly in all media tested as follows: the highest acrosome losses were observed in the fresh-phos and EDTA extenders without egg yolk; the lowest rate was observed with Tris-glucose supplemented egg yolk extender. In conclusion, at 4 degrees C, egg yolk extender best-protected sperm motility parameters. Differences in osmolarity between the extenders in terms of substrate related to sperm metabolic activity may explain the optimal results obtained using egg yolk-supplemented Tris-glucose extender, which preserved motility and acrosome integrity in chilled dog semen. These results indicated that good quality dog spermatozoa could be preserved for up to 10 d.     

Activated pericyte attenuates endothelial functions: nitric oxide-cGMP rescues activated pericyte-associated endothelial dysfunctions.

Hepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte-endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO-cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependent manner.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.     

In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen

Substitution of egg yolk with soybean lecithin may reduce hygienic risks in extenders. Though a few studies have been performed on the effect of soybean lecithin in bull, to date evaluation of ram semen in vitro fertility after cryopreservation with use of soybean lecithin has not been studied. This study assessed the effect of 1% or 2% (wt/vol) soybean lecithin (L1 or L2) or 15% or 20% (vol/vol) egg yolk (E15 or E20) supplemented with 5% or 7% glycerol (G5 or G7) in a Tris-based medium for cryopreservation of ram (Oviss arries) semen. Although no significant difference was observed in pattern of capacitation, the best results in terms of sperm motility, viability postthaw, and cleavage rates were observed with L1G7 (51.9 ± 4.8%, 48.1 ± 3.5%, and 79.6 ± 3.9%, respectively) and E20G7 (51.8 ± 2.9%, 46.7 ± 4.0%, and 72.9 ± 6.4%, respectively). Our results also showed that 1% lecithin and 20% egg yolk was superior to 2% lecithin and 15% egg yolk. In terms of cleavage rate, 7% glycerol was superior to 5% glycerol. No significant difference was obtained between groups in terms of blastocysts rate per cleaved embryo. Therefore, we concluded that the optimal concentration of lecithin and egg yolk is 1% and 20%, respectively, along with 7% glycerol. In addition, our results suggest that lecithin can be used as a substitute for egg yolk.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Antisense oligonucleotides modulate high glucose-induced laminin overexpression and cell proliferation: a potential for therapeutic application in diabetic microangiopathy

Vascular basement membrane (BM) thickening is a prominent and characteristic lesion of diabetic microangiopathy. Studies suggest that increased synthesis of laminin, a BM component, is associated with the development of thickened BM in diabetic vessels. In this study, we evaluated whether an interventive strategy using laminin antisense phosphorothioate oligonucleotides (Lam AS-oligos) could specifically inhibit high-glucose-induced laminin gene overexpression in vascular endothelial cells and normalize cell proliferation. Rat endothelial cells grown in high-glucose (30 mM) medium for 7 days showed increased laminin mRNA and protein level (195% +/- 28% of control, p < 0.05; 143% +/- 26% of control, p < 0.05, respectively) and reduced cell number (79% +/- 6% of control, p < 0.05) compared with cells grown in normal (5 mM) glucose medium. When cells grown in high-glucose medium were transfected with 0.4 microM Lam AS-oligos for 48 hours in the presence of 8 microM lipofectin, the laminin mRNA and protein level decreased (121% +/- 19% and 99% +/- 15% of control, respectively), and the cell number was restored to near normal level (93% +/- 7% of control). The results indicate that the antisense strategy is effective in selectively reducing laminin overexpression and improving endothelial cell proliferation under high-glucose conditions. Thus, the As-oligos may be potentially useful for preventing the development of thickened vascular BM in diabetic microangiopathy.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Hemopoiesis in spleen and bone marrow cultures

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics in vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.     

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Comparison of the intracellular lipids of cultured vascular endothelial cells and fibroblasts

Summary The lipids of human umbilical vein endothelial cells, calf aortic endothelial cells and foreskin fibroblasts have been compared. Cell cultures were established, and, upon confluency, the lipids were extracted and analyzed with respect to total lipid content, classes of lipids and total lipid fatty acid composition. The total quantity of lipid per milligram protein found in both human umbilical vein endothelium and calf aorta endothelium was similar to that found in fibroblasts grown in similar medium. Both types of endothelium contained the same major neutral lipid classes as fibroblasts, although they contained more phospholipid than did fibroblasts. The fatty acid composition of the three cells examined was influenced by cell type as well as the type of serum in the culture medium. This work was supported by PHS Grants HL16058, HL19638, AM14626 and HL18827.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development.

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.     

Primitive endothelial cell lines from the porcine embryonic yolk sac

Endothelial cell lines have been established from cells that were isolated from porcine yolk sacs from day 18 and day 22 embryos and propagated in vitro under various growth conditions. After expansion in vitro, the general properties of the cells proved similar for the different media used. The endothelial cells expressed cell surface receptors for acetylated low-density lipoprotein and also expressed cell surface-associated angiotensin-converting enzyme. The cells showed a characteristically high level of binding for Bandeiraea simplicifolia lectin I and Dolichos biflorus agglutinin but did not bind significant amounts of Ulex europaeus lectin I. The cells expressed low but serologically detectable levels of Class I major histocompatibility complex (MHC) antigens but failed to bind antibodies directed against Class II MHC antigens. Alpha5beta1 integrins were weakly expressed, whereas vascular cell adhesion molecule-1 (CD106) and alphavbeta3 integrins were not detected. Three-dimensional tube formation was readily observed in cultures grown on Matrigel and occurred even in uncoated plastic dishes in the absence of Matrigel. In contrast to most of the adult porcine endothelial cells, yolk sac-derived endothelial cells did not possess serologically detectable receptors for porcine growth hormone (GH), an observation consistent with the finding that GH did not increase the proliferative rate of these cells. Electron microscopic examination demonstrated the presence of Weibel-Palade bodies, tight endothelial cell junctions, and typical rough endoplasmic reticulum. Exposure of the cells to either concanavalin-A-stimulated porcine splenocyte culture supernatants or to human tumor necrosis factor alpha did not cause upregulation of VCAM-1 or Class II MHC antigens. Addition of porcine interferon-gamma led to an increase in the level of expression of Class I MHC. Yolk sac endothelial cells from day 22 embryos showed a low but detectable level of expression of Class II MHC antigens, whereas the endothelial cells from day 18 embryos showed no expression of Class II antigens after interferon-gamma stimulation. The cells maintained competence to develop vascular structures in vitro and could do so after coinjection with murine tumor cells into adult, immunocompromised mice.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.