Endothelial Metabolism Research Articles

Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor reduces proteoglycan synthesis in cultured endothelial cells.

Tumor necrosis factor (TNF)-induced disruption of vascular endothelial barrier function may be due in part to alterations in proteoglycan metabolism. To test this hypothesis, confluent endothelial cell monolayers were exposed for 24 h to 500 or 1,000 U of TNF per milliliter of culture medium together with 20 microCi Na2 35SO4. HPLC anion-exchange separation of proteoglycans secreted into media of control as well as TNF-treated cultures revealed one major peak (representing 95% of total radioactivity) and one minor peak (representing 5% of total radioactivity), which eluted at 0.6 and 0.9 M NaCl, respectively. One single peak was obtained from control as well as TNF-treated endothelial cell monolayers and eluted at 1.2 M NaCl. TNF treatment did not change the total quantity of radioactive proteoglycans secreted into the media but significantly decreased the amount of proteoglycans in endothelial cell monolayers. However, TNF treatment did not alter the size or glycosaminoglycan (GAG) composition of the proteoglycans either in the media or in the cell monolayers. In addition, mRNA levels of specific proteoglycans, perlecan and biglycan, were measured upon TNF treatment, using Northern analysis. TNF treatment caused a dose-dependent decrease in mRNA levels for the core proteins of perlecan, a major heparan sulfate proteoglycan (HSPG), and biglycan in endothelial cultures. These results suggest that TNF decreases production of proteoglycans and alters normal endothelial cell proteoglycan metabolism which may be sufficient to impair endothelial barrier function.

Effects of Sex Hormones on Cyclic Nucleotide Metabolism in Cultured Bovine Aortic Endothelial Cells

The effects of testosterone and estradiol on the regulation of the cyclic nucleotide metabolism in primary cultured endothelial cells from bovine aortae were examined. Testosterone added 24 h after plating increased the adenosine 3’,5’-cyclic monophosphate (cAMP) concentration from 20.3 to 40.6 pmol/mg protein, whereas the guanosine 3’,5’-cyclic monophosphate (cGMP) level decreased from 1.9 to 0.8 pmol/mg protein over a 5-day period. Estradiol increased cAMP from 20.3 to 30.3 pmol/mg protein and increased cGMP from 1.9 to 3.5 pmol/mg protein. The action of testosterone was potentiated by the addition of 10 mM theophylline. The basal level of the adenylate cyclase activity in control cells and the elevated level in treated cells were stimulated by sodium fluoride. In the presence of isobutylmethylxanthine, prostacyclin produced the most significant stimulation followed by isoproterenol and prostaglandin E1. In contrast, phenylephrine failed to stimulate the adenylate cyclase activity significantly. Testosterone added to subconfluent cultures at a low concentration (1 × 10–9M) failed to stimulate adenylate cyclase, but did inhibit low-Km phosphodiesterase (p < 0.05). A high concentration (1 × 10–7M) of testosterone stimulated the adenylate cyclase activity, markedly decreased the activities of high- and low-Km phosphodiesterase, and caused a reduction in matrix fibronectin, rearrangement of actin, and a change in cell surface topography. This study showed that testosterone induced a major change in endothelial cell cyclic nucleotide metabolism by modulating the activities of adenylate cyclase and cAMP phosphodiesterase, whereas estradiol induced a major change in cGMP metabolism.

The role of endothelium in the pathogenesis of diabetic microangiopathy.

Damage caused to the vessel wall by diverse mechanisms may lead to diabetic microangiopathy. Consequently, research work is more and more focusing on the pathophysiology of vascular cells, with particular emphasis on endothelium. This paper reviews the present knowledge on the alterations of small vessel endothelium in diabetes. The most important risk factors for diabetic microangiopathy are the duration of disease and the degree of metabolic control maintained throughout the years. However, genetic factors may also contribute. These are examined first, followed by the presumed roles played by increased protein glycation and the production of Advanced Glycosylation End Products, the "polyol pathway" and free radical generation. Endothelium is a widespread, extremely active organ which regulates complex physiologic functions and its structure and function are discussed in the second section of this review. The third part deals with how diabetes can affect endothelium and describes observations on endothelial metabolism in vitro as well as morphologic and functional alterations in the patients. Unfortunately, the mechanisms leading to progressive degeneration of the microcirculation and organ damage in diabetic patients remain largely unaccounted for.

Endothelium-dependent contractions to oxygen-derived free radicals in the canine basilar artery.

Experiments were designed to determine the effect of oxygen-derived free radicals in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution bubbled with 95% O2-5% CO2 (temperature = 37 degrees C; pH = 7.4). A radioimmunoassay technique was used to measure production of prostaglandins and thromboxane B2. Xanthine oxidase (1-9 mU/ml, in the presence of 10(-4) M xanthine) and hydrogen peroxide (10(-6) to 10(-4) M) caused concentration-dependent contractions. The removal of endothelium reversed these contractions into relaxations. Contractions to xanthine oxidase and hydrogen peroxide were inhibited in the presence of superoxide dismutase (150 U/ml), catalase (1,200 U/ml), indomethacin (10(-5) M), and SQ 29548 (10(-6) M) but not in the presence of deferoxamine (10(-4) to 10(-3) M) and dimethyl sulfoxide (10(-4) M). NG-monomethyl-L-arginine (3 x 10(-5) M) augmented the contractions to hydrogen peroxide. Xanthine oxidase stimulated production of 6-ketoprostaglandin F1 alpha, prostaglandin F2 alpha, prostaglandin E2, and thromboxane B2. The stimulatory effect was prevented by the removal of endothelial cells. These studies suggest that xanthine oxidase causes endothelium-dependent contractions mediated by: 1) hydrogen peroxide-induced stimulation of the endothelial metabolism of arachidonic acid via the cyclooxygenase pathway, leading to activation of prostaglandin H2-thromboxane A2 receptors, and 2) inactivation of basal production of nitric oxide by superoxide anions.

Comprehensive model of transport and metabolism of adenosine and S-adenosylhomocysteine in the guinea pig heart.

Regulation of blood flow and mitochondrial respiration in the heart would be clarified by improved knowledge of interstitial concentrations and cellular production rates of adenosine; however, these variables cannot be measured directly. To interpret indexes that are available, a comprehensive mathematical model was developed, based on a large body of experimental data. The model describes most of the important pathways of capillary-tissue transport and cellular metabolism of adenosine in the guinea pig heart. It includes capillary flow, solute transport between tissue regions, nonlinear enzyme kinetics for adenosine kinase and adenosine deaminase, and reversible biunireactant kinetics for S-adenosylhomocysteine hydrolase in cardiomyocytes and endothelial cells, intracellular production of adenosine via AMP hydrolysis and transmethylation, and extracellular production of adenosine. A single set of parameter values for the model was obtained in the first stage of the analysis by taking certain values directly from published sources, other values were subject to specific constraints, and other values were determined by parameter optimization. The effects of flow and endothelial metabolism on the relation between interstitial and venous adenosine concentrations were determined. The relation between myocardial adenosine production rate and S-adenosylhomocysteine accumulation in the presence of excess homocysteine was estimated. In the second stage of the analysis, the model was used to investigate the mechanism of myocardial adenosine production, without changing the parameter values. Cellular adenosine production rates were estimated by fitting measurements of venous adenosine release obtained during altered energetic conditions in experiments by different investigators. The original results showed a dissociation between measurements of cytosolic AMP concentrations and venous adenosine release. It is concluded that 1) it is essential to account for the effect of flow on interstitial and venous adenosine concentrations, since decreased flow may produce effects outwardly resembling inhibition of the enzyme 5'-nucleotidase, 2) adenosine concentrations in epicardial transudate are not in equilibrium with interstitial fluid, and 3) the rate of cellular adenosine production increases monotonically with free cytosolic concentrations of AMP during a variety of alterations in energy balance of the guinea pig heart.

Hypoxia induces glucose transporter expression in endothelial cells.

Endothelial cells in various tissues of the body are often exposed to hypoxic conditions. To examine the effects of sustained hypoxia on energy metabolism in endothelial cells, we have maintained bovine aortic and human umbilical vein endothelial cells in an atmosphere containing low oxygen concentrations (14 mmHg) for up to 96 h. We report here that endothelial cells maintained under these conditions upregulate their glucose transport activity, consume more glucose, and produce greater amounts of lactic acid than normoxic cells. Upregulation of glucose transport activity by hypoxic endothelial cells required several hours to occur, was associated with increased expression of mRNA and protein for the erythroid/brain form of the facilitative glucose transporter, and was not due to depletion of glucose from the medium. Prolonged treatment of endothelial cells with inhibitors or uncouplers of oxidative phosphorylation (antimycin, azide, dinitrophenol) under normoxic conditions also upregulated glucose transporter expression. These results suggest that reduced rates of oxidative metabolism may represent an important signal for cells to adapt metabolically to hypoxia. Furthermore, in our examination of endothelial cell energy metabolism, we discovered that endothelial cells contain phosphocreatine and express both the brain and muscle isozymes of creatine kinase.

The regulation of endothelin production in human umbilical vein endothelial cells: unique inhibitory action of calcium ionophores.

Endothelins (ETs) are a recently discovered family of small proteins that have potent long-lasting vasoconstrictive activities. Increased circulating concentrations of ETs have been found in hypertensive and renal disorders, including pregnancy-induced hypertension (PIH). PIH has been postulated to be the end result of endothelial cell damage and aberrant calcium metabolism. We evaluated the effects of calcium ionophores, calcium channel blockers, and two forms of cellular damage on ET production by human umbilical vein endothelial cells (HUVEC). Cells were grown to confluence and then incubated for 16 h with these treatments: physical trauma ("scratching"), oxidant damage (hydrogen peroxide, 1-20 mM), ionomycin (0.25-2.0 microM), A-23187 (10(-9)-10(-5) M), verapamil (0.22-22.0 microM), and nifedipine (2-200 micrograms/mL). ET production was determined using a commercial RIA that detects ET-1 and ET-2. Physical trauma enhanced ET production, whereas oxidant damage had the opposite effect. Both ionomycin and A-23187 caused concentration-dependent inhibition of ET production. Neither verapamil nor nifedipine consistently altered ET production. We conclude that specific forms of cellular damage can stimulate HUVEC ET production, although oxidant damage may be slightly inhibitory. Thus, enhanced ET levels in PIH may represent endothelial cell activation, rather than damage. HUVEC ET production is regulated in an inverse manner by intracellular calcium concentrations, suggesting a negative feedback from mediators of ET action on cells.

Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils.

There is increasing evidence that the glycosaminoglycan (GAG) component of the vascular endothelium is important in regulating vascular permeability, thromboresistance and cellular interactions. We have investigated the GAG metabolism of cultured human umbilical vein endothelial cells (HUVEC) in response to a range of inflammatory stimuli. Using both chemical measurement of cellular and supernatant GAGS and 35S labelling to identify newly synthesised GAGS, interleukin 1 (IL1), tumour necrosis factor (TNF) and interferon gamma (IFN gamma) were shown to influence sulphated GAG metabolism significantly. IL1 and TNF caused a marked increase in culture supernatant GAGS and a concomitant reduction in cell-associated GAGS. This was shown histochemically to be associated with a marked reduction and redistribution of endothelial surface anionic sites. The addition of neutrophils to HUVEC pretreated with Escherichia coli endotoxin, IL1 or TNF resulted in a further reduction in both cellular GAGS and surface anionic sites. These results suggest that changes in endothelial cell GAG metabolism during inflammation may contribute to the disturbance of vascular endothelial homeostasis associated with infectious and inflammatory states.

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-α

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O 2 − and H 2O 2, in endothelial cells. Then we examined the influence of tumor necrosis factor-α and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O 2 − and H 2O 2. We determined a basal intracellular H 2O 2 concentration of 32.8 ± 10.7 p m in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 ± 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 ± 3.79 mU/ mg protein. Only 0.48 ± 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H 2O 2 release was 75 ± 12 pmol · min −1 · mg cell protein −1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 ± 0.75 μU/mg with 47.1 ± 11.7% in the oxidase form. Intracellular purine levels of 1.19 ±1.04 nmol hypoxanthine/mg protein, 0.13 ± 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/ oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 μg/ml lipopolysaccharide (LPS) for 1–12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H 2O 2 in the direct endothelial toxicity of TNF and LPS.

Effects of indomethacin on PMA-induced pulmonary endothelial enzyme dysfunction in vivo.

We investigated the effects of phorbol myristate acetate (PMA) on metabolic pulmonary endothelial ectoenzyme dysfunction. Anesthetized rabbits were placed on total heart bypass, and the single-pass transpulmonary metabolism of [3H]benzoyl-Phe-Ala-Pro (BPAP) by endothelial-bound angiotensin-converting enzyme (ACE) and [14C]adenosine 5'-monophosphate (AMP) by 5'-nucleotidase (NCT) was calculated before and after PMA (10 micrograms/kg iv), a dose that does not produce histologically evident endothelial damage. Under conditions of partial microvascular recruitment (blood flow = 400 ml/min through the entire lung), PMA, but not the vehicle, significantly reduced substrate utilization of both BPAP and adenosine 5'-monophosphate (AMP) and increased the apparent Michaelis constant (Km) values of ACE for BPAP, indicative of metabolic dysfunction. These changes were completely prevented by pretreatment with indomethacin. Under conditions of near full microvascular recruitment (blood flow = 640 ml/min through the left lung only), PMA similarly reduced substrate utilization and increased the apparent Km of ACE for BPAP. In this case, however, indomethacin failed to prevent the observed PMA-induced metabolic dysfunction. We conclude that PMA alters endothelial ectoenzyme substrate metabolism independently from changes in pulmonary blood flow; indomethacin appears to antagonize the effects of PMA under conditions of partial microvascular recruitment only, perhaps by diverting flow to previously unperfused, unexposed to PMA, and hence metabolically healthy vessels.

Human platelet-derived transforming growth factor-beta stimulates synthesis of glycosaminoglycans in cultured porcine aortic endothelial cells.

Human platelet-derived transforming growth factor-beta (TGF-beta) stimulated the incorporation of [35S]sulfate into glycosaminoglycans (GAGs) both in the medium and on the cell surface of cultured aortic endothelial cells in a dose- and time-dependent manner. TGF-beta inhibited the suppression of GAG synthesis by other cytokines. TGF-beta increased the protein synthesis, while reducing the DNA synthesis by endothelial cells. The proportion of anticoagulant heparan sulfate in total GAGs was reduced. Thus, TGF-beta affected the endothelial GAG metabolism both quantitatively and qualitatively.

Diethylcarbamazine inhibits endothelial and microfilarial prostanoid metabolism in vitro

Diethylcarbamazine (DEC) rapidly lowers the number of microfilariae in the peripheral circulation. The mechanism of action is unknown, but may involve alterations of arachidonic acid metabolism in vascular tissues. We studied the effects of DEC on arachidonic acid metabolism by bovine pulmonary arterial endothelium monolayers, human platelets and Brugia malayi microfilariae. DEC at a concentration of 2.5 microM, a level achieved in vivo, rapidly decreased prostacyclin, prostaglandin E2 and thromboxane B2 release from endothelial monolayers by 78% (P less than 0.001), 57% (P = 0.05), and 75% (P less than 0.05), respectively. High-pressure liquid chromatography of extracts of endothelial monolayers incubated with DEC showed similar inhibition of these cyclooxygenase pathway products, but exposure to the drug did not result in formation of new eicosanoids. DEC did not inhibit endothelial phospholipase A2-dependent release of arachidonate from membrane stores, whereas prostaglandin H2 synthase activity (cyclooxygenae, EC 1.14.99.1) was reduced to a degree similar to that effected by acetylsalicylic acid. Microfilarial but not platelet synthesis of cyclooxygenase products was also reduced by DEC. These data suggest that the mechanism by which DEC lowers the level of microfilariae in the circulation may in part involve its effects on host endothelial and parasite eicosanoid production.

Membrane fatty acid composition and endothelial cell functional properties

In order to study the influence of endothelial cell fatty acid composition on various membrane related parameters, several in vitro methods were developed for manipulating the fatty acid content of human endothelial cell membranes. Changes in membrane fatty acid profile were induced by using fatty acid modified lipoproteins or free fatty acids. The largest changes in endothelial fatty acid composition were obtained by culturing the cells in media supplemented with specific free fatty acids. An increase in arachidonic acid content of endothelial phospholipids was induced by supplementation with saturated fatty acids or with arachidonic acid itself. A decrease in arachidonic acid content was obtained by supplementation with other unsaturated fatty acids. Under the experimental conditions used endothelial cells showed a low desaturase activity and a high elongase activity. Considerable alterations in membrane fatty acid composition did not greatly influence certain membrane related parameters such as polymorphonuclear leukocyte adherence and endothelial cell procoagulant activity. In general, for fatty acid modified endothelial cells an association between endogenous arachidonic acid content and total production of eicosanoids was found. This study demonstrates that considerable changes in membrane fatty acid profile affect endothelial cell arachidonic acid metabolism, but it also illustrates homeostasis at the level of endothelial cell functional activity.

Differences in prostaglandin metabolism in cultured aortic and pulmonary arterial endothelial cells exposed to acute and chronic hypoxia.

In vivo, a marked difference in blood oxygen tension exists between the pulmonary artery and the aorta. Responses of vascular endothelial cells from these vessels to changes in ambient oxygen might be influenced by the oxygen tension to which they are continuously exposed in vivo or by their anatomic site. To explore this hypothesis, we initially studied the production of the cyclooxygenase metabolites prostacyclin and thromboxane in bovine aortic and main pulmonary arterial endothelial cells grown in 21% O2 and exposed to different degrees of acute hypoxia over a wide range of times. We found that short-term hypoxia (3% or 0% O2) rapidly and transiently activates the cyclooxygenase pathway in both cell types, with a more rapid response in bovine aortic endothelial cells. To determine whether culture in an oxygen tension similar to that to which main pulmonary arterial endothelial cells are exposed in vivo alters this response, we evaluated these cyclooxygenase metabolites in bovine aortic and main pulmonary arterial endothelial cells cultured long-term in 3% O2, both at baseline and after exposure to acute anoxia (0% O2). In both cell types, we found a decrease in prostacyclin and thromboxane synthesis at baseline and evidence of an increase in the Vmax of thromboxane synthetase following stimulation with exogenous arachidonic acid. In chronically hypoxic cells exposed to acute anoxia, there were marked differences in enzyme activity compared with that in endothelial cells maintained in 21% O2 with differences depending on the origin of the endothelial cells. In bovine aortic endothelial cells, production of neither cyclooxygenase metabolite increased; in bovine main pulmonary arterial endothelial cells, only thromboxane production increased, suggesting isolated activation of the cyclooxygenase-thromboxane synthetase pathway. These studies demonstrate that acute and chronic hypoxia have profound effects on endothelial cell cyclooxygenase metabolism and that these effects depend on the duration and degree of the hypoxic exposure and the vascular bed from which the endothelial cells are derived.

Endothelial purine metabolism: A role in regulating vasomotion?

Endothelial purine metabolism: A role in regulating vasomotion?

Energetic response of coronary endothelial cells to hypoxia

The response of endothelial energy metabolism to oxygen supply was studied in cultured coronary endothelial cells from the rat at defined PO2 levels between 0.1 and 100 Torr. In the presence of glucose (5 mM), endothelial respiration (4 nmol O2.min-1.mg protein-1) was independent of the exterior PO2 greater than 3 Torr; oxygen consumption was half maximal at 0.8 Torr. At 100 Torr, lactate production was 26 nmol.min-1.mg protein-1; the decrease of the PO2 to 0.1 Torr resulted in a 2.2-fold increase in lactate production. The contents of ATP, ADP, and AMP were 21, 4, and 2 nmol/mg protein, respectively; they remained constant for 2.5-h incubations at PO2 levels between 0.1 and 100 Torr. In the presence of palmitate (100 microM) plus glutamine (0.5 mM), oxygen consumption was 8 nmol.min-1.mg protein-1 at PO2 levels greater than 3 Torr, and the half-maximal rate was again observed at 0.8 Torr. Lactate production was negligible. At PO2 levels greater than 3 Torr, the cells remained well energized. Below 3 Torr, however, the adenine nucleotide contents rapidly declined. These results demonstrate that the oxygen demand of coronary endothelial cells is low compared with the beating myocardium. In the presence of glucose, aerobic glycolysis is pronounced and the Pasteur effect small. In severe hypoxia (PO2 less than 0.1 Torr) the energetic state remained stable. In the absence of glucose, the energetic state of coronary endothelial cells is sensitive to the exterior PO2 less than 3 Torr, declining concomitantly with the decrease in respiration.

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E 1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 μM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E 1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine, which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.

Influence of natural and recombinant interleukin 2 on endothelial cell arachidonate metabolism. Induction of de novo synthesis of prostaglandin H synthase.

We studied the effects of natural and recombinant human IL-2 (rIL-2) on secretion of prostacyclin (PGI2), vWf, and tissue-type plasminogen activator (tPA). IL-2 elicited a steady increase in PGI2 synthesis by cultured human umbilical vein endothelial cells (HUVECS) and bovine aortic endothelial cells but had no effect on vWf or tPA. Both purified natural IL-2 (nIL-2) and rIL-2 induced significant PGI2 synthesis. Substitution of the cysteine residue at position 125 of rIL-2 with serine or alanine led to loss of PGI2-stimulatory activity in HUVECS without affecting thymidine incorporation in lymphocytes. HPLC analysis of arachidonate metabolites detected predominantly 6 keto-PGF1 alpha (6KPGF1 alpha) peak. Treatment of cultured endothelial cells with cycloheximide and actinomycin D resulted in inhibition of 6KPGF1 alpha synthesis. The Western blot using a polyclonal antibody against PGH synthase revealed an increment in the 70-kD subunit of PGH synthase by nIL-2 and rIL-2, but not by alanine-substituted rIL-2. We conclude that IL-2 stimulated sustained PGI2 production by a mechanism that includes the de novo synthesis of PGH synthase. This mechanism for regulating AA metabolism probably has important physiologic implications.

Intralipid effect on normal and hypoxic remodeled rat pulmonary vasculature.

Intralipid (IL) infusion has been associated with pulmonary vasoconstriction and decreased oxygenation and may worsen preexisting pulmonary vascular changes. To investigate this, we infused IL or 0.9% saline for 1 wk in normal Sprague-Dawley rats and in rats with vascular changes induced by a previous 2-wk exposure to chronic hypobaric hypoxia (air at 380 mmHg). At postmortem we quantitatively evaluated arterial changes in the left lung by light microscopy and alterations in endothelial cells in the right lung by electron microscopy. In rats maintained in room air, 1-wk IL infusion resulted in extension of muscle into alveolar wall and duct arteries (P less than 0.001 for both), medial hypertrophy of arteries 50-99 microns external diameter (P less than 0.01), reduced arterial density (P less than 0.05), and an increase in volume density of endothelial smooth endoplasmic reticulum (P less than 0.05). In post-hypoxia rats, however, IL infusion did not induce further progression of the more severe arterial and endothelial changes observed. To determine whether IL-associated vascular abnormalities may be related to vasoconstriction, different rats were instrumented under pentobarbital sodium anesthesia with indwelling cardiovascular catheters and, 2 days later, with the animals fully conscious, the hemodynamic response assessed. An acute 15-min IL infusion caused a significant increase in pulmonary artery pressure and mild hypoxemia (P less than 0.05 for both) in room air rats but not in the posthypoxia group. This response was not, however, sustained over a 2-day IL infusion. Thus IL induces pulmonary vascular abnormalities that do not appear to be related to vasoconstriction or hypoxemia. We speculate that differences in endothelial metabolism of IL in room air and posthypoxia rats may explain the lack of IL-related abnormalities in the latter group.