Human dermal microvascular endothelial cells in vitro: Effect of cyclic AMP on cellular morphology and proliferation rate

Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitor. In the presence of 5 x 10^?4 M dibutyryl cyclic AMP (Bt₂cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of control values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt₂cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt₂cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methyl-xanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt₂cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt₂cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.     

Microvessel endothelial cell transdifferentiation: phenotypic characterization

Human dermal microvessel endothelial cells (MEC) have two basic functions: maintenance of tissue homeostasis and facilitation of inflammatory responses. The former requires that the endothelium expresses traits of an epithelium, while inflammatory reactions are associated with intimal disruption. Acute inflammation transiently alters endothelium, whereas chronic inflammation may result in vessel reorganization and MEC mesenchymalization. Foreskin MEC in vitro undergo a similar epithelial-mesenchymal modulation. In the presence of cAMP, cultivated dermal MEC exhibit the structural and functional characteristics of an epithelium. MEC grown in cAMP-deficient medium initially have a "transitional" configuration and are subsequently transformed into mesenchymal cells. If cAMP is replaced by histamine, MEC maintain a stable intermediate transitional configuration. Transitional MEC refed cAMP-supplemented medium revert to an epithelial phenotype, whereas parallel cultures fed cAMP-deficient medium are transformed into mesenchymal cells. Phenotypic modulation can be induced without cell division and thus provides a unique example of direct transdifferentiation. Our data furthermore suggest that this transdifferentiation results in the acquisition of properties usually attributed to cells of the reticuloendothelial system.     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors

Summary Human dermal microvascular endothelial cells are used to analyze the functions of microvascular endothelium in vitro. However, the low yield and short lifespan of these cells in culture has limited the types of analysis that could be performed. Human microvascular endothelial cells are typically grown in media containing supplements such as dibutyryl cyclic AMP, hydrocortisone, bovine brain extract, and antifungal agents, each of which increase the complexity of experimental design and interpretation of results. In the present study, endothelial cells were transferred after Ulex europeus-I selection into a simplified medium consisting of Endothelial Basal Medium 131, 10% fetal bovine serum, and 2 ng/ml basic fibroblast growth factor and analyzed over 3 mo. The human microvascular endothelial cells expressed the endothelial markers von Willebrand factor, CD31, P-selectin, and E-selectin. In addition, the cells showed increased proliferation in the presence of basic fibroblast growth factor (0.5 ng/ml) or vascular endothelial cell growth factor (10 ng/ml). Tumor necrosis factor-α-induced expression of E-selectin was similar in cells at Passages 3, 6, and 12, indicating that the cells maintained responsiveness to cytokines over several weeks. Furthermore, the endothelial cells attained a typical cobblestone morphology with increased cellular density and also formed capillarylike tubes on Matrigel. In summary, the human dermal microvascular endothelial cells display the expected endothelial characteristics when grown for several passages and, therefore, provide a valuable in vitro model for human microvascular endothelium.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Possible role of adenosine cyclic 3':5'-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6,O2-dibutyryl adenosine cyclic 3':5"-monophosphate.

We have demonstrated that in Chinese hamster ovary (CHO) cells, N6,O2'-dibutyryl adenosine cyclic 3':5'-monophosphate (dibutyryl cyclic AMP) has a remarkable morphogenetic effect in converting cells of a compact, epithelial-like morphology into a spindle-shaped, fibroblast-like form. Homogenates of CHO cells were found to contain two adenosine cyclic 3':5'-monophosphate (cyclic AMP) phosphodiesterase (EC 3.1.4.c) activities, which differ in apparent Km with respect to their substrate, cyclic AMP. These were designated cyclic AMP phosphodiesterase I, with a low Km of 2 to 5 muM and cyclic AMP phosphodiesterase II, with a high Km of 1 to 3 mM. Cyclic AMP phosphodiesterase I was competitively inhibited by N6-monobutyryl and dibutyryl cyclic AMP, with apparent Ki values of 40 to 60 muM and 0.25 to 0.35 mM, respectively. Experimental evidence demonstrates that the effect of exogenous dibutyryl cyclic AMP on cell morphology is a result of an increase in the endogenous level of cyclic AMP. This increase appears to be due largely to the inhibitory action of intracellular N6-monobutyryl cyclic AMP on cyclic AMP phosphodiesterase I, which results in a decreased rate of degradation of intracellular cyclic AMP.     

Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine

We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells. Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes. Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity. Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values. Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value. After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen. In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250%. 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled. The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol. In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity. The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol. On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures. It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.     

Effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro: dependence of the effect on N6,O2'-dibutyryl Adenosine 3',5'-cyclic monophosphate.

The inhibitory effect of dithiothreitol on meiotic maturation of mouse oocytes in vitro is a function of the intracellular dibutyryl cyclic AMP concentration. Inhibition of nuclear (germinal vesicle) breakdown by dibutyryl cyclic AMP is reversed upon transfer of oocytes to plain culture medium, whereas, transfer to medium containing dithiothreitol results in continued inhibition. Dithiothreitol significantly enhances the effectiveness of dibutyryl cyclic AMP as an inhibitor of meiotic maturation. These results suggest that the reported effect of sulfhydryl reducing agents on membrane dissolution and reconstitution, in both meiotic and mitotic cells, may be attributed to their influence on intracelular levels of cyclic AMP.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Dissociation of the Stellate Morphology from Intracellular Cyclic AMP Levels in Cultured Rat Brain Astroglial Cells: Effects of Ganglioside GMl and Lysophosphatidylserine

Secondary microcultures of newborn rat cerebrum astroglial (AG) cells, maintained in a serum-free, chemically defined medium, were treated with various agents known to elevate intracellular cyclic AMP (cAMP) levels. Earlier studies had shown these drugs to induce a process-bearing (stellate) morphology in the AG cells, a response that was antagonized by the presence of gangliosides. One millimolar dibutyryl cyclic AMP (dBcAMP), 10 microM forskolin, 12 nM cholera toxin, and 30 microM isoproterenol all raised intracellular cAMP levels, from basal values of 3 pmol/10(6) cells to 30-30,000 pmol/10(6) cells, depending on the agent tested. dBcAMP caused the greatest elevation, and forskolin the least. The timing and/or the level of the AMP response did not precisely correlate with those of the stellation response. Values of ED50 with the four agents, as determined for the cAMP response, were always higher than stellation ED50 values in all treatments, and ED50 did not correlate with the maximal levels of cyclic AMP induced by the four agents. The capacity of ganglioside GM1 to block the stellation response to the four agents was not accompanied by a similar capacity to block the cAMP responses. Lysophosphatidylserine (lysoPS) had the capacity to induce AG cell stellation as well, without altering the basal level of cAMP. Both lysoPS and gangliosides, therefore, may act directly on the cellular machinery underlying the stellation response without involving changes in intracellular AMP.     

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase. However, at higher concentrations of dibutyryl cyclic AMP there is G1 cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphocyte activation may occur by different mechanisms that are dose dependent.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Collagen I initiates endothelial cell morphogenesis by inducing actin polymerization through suppression of cyclic AMP and protein kinase A

Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha(1)beta(1) and alpha(2)beta(1), followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords.     

A variant of S49 mouse lymphoma cells with enhanced secretion of cyclic AMP.

A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2'-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.