Stimulated Prostacyclin Production Research Articles

Low-affinity insulin-like growth factor binding protein 7 and its association with pulmonary arterial hypertension severity and survival.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of growth factor modifiers, some of which are known to be independently associated with pulmonary arterial hypertension (PAH) survival. IGFfactor binding protein 7 (IGFBP7) is a unique low-affinity IGFBP that, independent of IGF, stimulates prostacyclin production. This study proposed to establish associations between IGFBP7 and PAH severity and survival, using enrollment and longitudinal samples. Serum IGFBP7 levels were significantly elevated in patients with PAH compared to controls. After adjusting for age and sex, logarithmic increases in IGFBP7 were associated with a 20 m shorter six-minute walk distance (6MWD; p < 0.001), a 2-3 mmHg higher mean right atrial pressure (p < 0.001 and 0.02), and a higher likelihood of a greater REVEAL 2.0 risk category placement (p < 0.001). Kaplan-Meier analysis demonstrated significantly decreased survival with IGFBP7 above the median and Cox multivariable analysis adjusted for age and sex, demonstrated higher serum IGFBP7 was an independent predictor of survival. Though the exact mechanism is still unknown, given IGFBP7's role as a prostacyclin stimulant, it has potential use as a therapeutic target for disease modulation.

Abstract 13688: Low-Affinity Insulin-Like Growth Factor Binding Protein 7 and Its Association With Pulmonary Arterial Hypertension Severity and Survival

Introduction: Insulin like growth factor binding proteins (IGFBPs) are a family of growth factor modifiers, some of which are known to be independently associated with PAH survival. IGFBP7 is a unique low-affinity IGFBP that, independent of IGF, stimulates prostacyclin production. Hypothesis: Elevated IGFBP7 is associated with worse disease severity and survival in PAH. Methods: Using enzyme-linked immunosorbent assays (ELISA), we evaluated serum IGFBP7 in a multicenter PAH cohort, the NHLBI PAHBiobank (N=2583), and healthy controls (N=99). Hemodynamic and functional measures were used to assess IGFBP7’s association to PAH severity and survival. Kruskal Wallis was used for comparison and Spearman’s rank correlation for correlations. Linear and logistic regressions performed for associations. IGFBP7 was dichotomized at the median and analyzed by Kaplan-Meier survival and Cox proportional Hazard regression. Results: Serum IGFBP7 levels were significantly elevated in patients with PAH compared to controls. After adjustment, logarithmic increases in IGFBP7 were associated with a 38 meter shorter six-minute walk distance (6MWD; -37.7, 95%CI -54 to -21, p=&lt;0.001), a higher mean right atrial pressure (2.18mmHg, 95%CI 1.51-2.89, p&lt;0.001) and a higher REVEAL risk score (1.10, 95%CI 0.88-1.33, p &lt;0.001). Logarithmic increases in IGFBP7 were also associated with a two-times higher likelihood of having PAH associated with Portopulmonary disease (2.39, 95%CI 1.95-2.84, p&lt;0.001). Kaplan Meier analysis demonstrated significant survival effects of IGFBP7 above the median (see Figure 1) and an adjusted (age, sex, functional class, and 6MWD) Cox proportional hazards model (HR 3.1, 95% CI 2.2-4.4, p&lt;0.001). Conclusions: Higher circulating IGFBP7 levels were significantly associated with worse PAH severity and decreased survival. Given its role of as a prostacyclin stimulant, IGFBP7 may also serve as a therapeutic target for disease modulation.

Effects of the Catechol and Methoxy Metabolites of 17β-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2β, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2β metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2β metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2β metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2β metabolite-induced NO production. E2β and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-β antagonist) inhibited the rises in NOx levels stimulated by E2β and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (β2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2β, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2β metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy.

Comparative effects of immediate-release and extended-release aspirin on basal and bradykinin-stimulated excretion of thromboxane and prostacyclin metabolites.

A goal of aspirin therapy is to inhibit thromboxane production and platelet aggregation without inhibiting endothelial production of the vasodilator and anti‐thrombotic prostacyclin. This study tested the hypothesis that extended‐release aspirin (NHP‐554C) would have increased selectivity for inhibition of basal and simulated thromboxane formation compared to immediate‐release aspirin (ASA). Thirty‐six healthy subjects were randomized to NHP‐554C or ASA groups. Within each group, subjects were randomized to 5‐day treatment with 81 mg/d, 162.5 mg/d and placebo in a crossover design in which treatment periods were separated by 2‐week washout. On the fifth day of treatment, 81 mg/d and 162.5 mg/d ASA reduced basal urinary excretion of the stable thromboxane metabolite 11‐dehydro‐thromboxane B2 62.3% and 66.2% and basal excretion of the stable prostacyclin metabolite 2,3‐dinor‐6‐keto‐PGF1α 22.8% and 26.5%, respectively, compared to placebo. NHP‐554C 81 mg/d and 162.5 mg/d reduced 11‐dehydro‐thromboxane B2 53% (P = 0.03 vs. ASA 81 mg/d) and 67.9% and 2,3‐dinor‐6‐keto‐PGF1α 13.4% and 18.5%, respectively. NHP‐554C 81 mg/d did not significantly reduce basal excretion of the prostacyclin metabolite. Both doses of ASA and NHP significantly reduced excretion of both thromboxane and prostacyclin metabolites following intravenous bradykinin. During NHP‐554C 162.5 mg/d, but not during ASA, bradykinin significantly increased urinary 2,3‐dinor‐6‐keto‐PGF1α. Nevertheless, 11‐dehydro‐thromboxane B2 and 2,3‐dinor‐6‐keto‐PGF1α responses to bradykinin were statistically similar during ASA and NHP‐554C. In conclusion, at doses of 81 and 162.5 mg/d immediate‐ and extended‐release aspirin selectively decrease basal thromboxane production. Both forms of aspirin decrease bradykinin‐stimulated thromboxane and prostacyclin production, but some stimulated prostacyclin production remains during treatment with NHP‐554C.

Intracellular free calcium content plays a role in low magnesium-induced increases in prostacyclin production

In a previous study, we found that magnesium deficiency stimulated prostacyclin production and suggested that this stimulation resulted from an enhanced Ca2+ influx induced by magnesium deficiency. In this study, we further examined prostacyclin generation in cultured human umbilical vein endothelial cells after intracellular free calcium content ([Ca2+]i) was altered by addition of diltiazem, nifedipine, verapamil, sodium cyanide (NaCN), ruthenium red or quinidine to a low magnesium medium. The results showed that diltiazem, nifedipine, verapamil and ruthenium red inhibited 45Ca2+ influx, and NaCN and quinidine had no effect on 45Ca2+ influx. However, all of these compounds decreased [Ca2+]i, [3H]arachidonic acid release and prostacyclin production. The reduced [3H]arachidonic acid content in cellular phospholipids caused by low magnesium treatment was not altered by the added compounds. We suggested that arachidonic acid release and prostacyclin production was calcium-dependent in cultured endothelial cells.

Glycated type 1 collagen induces endothelial dysfunction in culture

Hyperglycemia-induced protein glycation is thought to be implicated in the diabetic vasculopathy. In this study, we cultured vascular endothelial cells on native or glycated collagen matrix and compared their growth and functional characteristics. At lower plating density, the cells grew equally well on both substrata; however, at higher planting density, the cells plated on glycated collagen grew slower and reached a lower confluent density compared to that of the native collagen-based cultures. Confluent cell layers formed on glycated collagen exhibited a lower diffusion barrier function and a less response to epidermal growth factor stimulated prostacyclin production, compared to their native collagen-cultured counterparts.

Tetrandrine and thapsigargin release arachidonic acid from cells in culture and stimulate prostacyclin production in rat liver cells, but may do so by different pathways

BackgroundTetrandrine inhibits tumor cell proliferation and demonstrates chemoprevention in cancer models. Speculation on the association between its effects on K+ and Ca2+ channels and cancer chemoprevention has been made. Thapsigargin also affects K+ and Ca2+ conductance. Thapsigargin, however, is a weak tumor promoter in the two-stage model of mouse skin carcinogenesis, yet it can induce apoptosis in androgen-independent prostatic cancer cells. I have postulated that arachidonic acid release from cells in culture is associated with cancer chemoprevention. The effects of tetrandrine and thapsigargin on arachidonic acid release from human colon carcinoma and rat liver cells and prostacyclin production by rat liver cells are compared in the current studies.ResultsTetrandrine and thapsigargin stimulate arachidonic acid release from human colon carcinoma and rat liver cells and prostacyclin production in rat liver cells. The stimulation by tetrandrine is not affected by incubation with actinomycin D, 100 mM KCl, the [Ca2+]i chelator, 1,2-bis (o-amino-5-fluorophenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethylester (BAPTA/AM) or in the absence of extracellular Ca2+. In contrast, stimulation by thapsigargin is inhibited by incubation with actinomycin D, 100 mM KCl, BAPTA/AM or in the absence of extracellular Ca2+.ConclusionBoth tetrandrine and thapsigargin stimulate arachidonic acid release, but based on the different results obtained in the presence of actinomycin D, the [Ca2+]i chelator, 100 mM KCl and in the absence of extracellular Ca2+, the mechanisms leading to this release and pathways leading to apoptosis and/or cancer chemoprevention may be different. Stimulations by tetrandrine may be mediated by activation of a secretory phospholipase A2, whereas thapsigargin's stimulations may be mediated by the cytoplasmic Ca2+-dependent phospholipase A2.

Sevoflurane but not isoflurane can reduce prostacyclin production of endothelial cells.

Little is known about the interaction of newer volatile anaesthetics with endothelial eicosanoid production. Sevoflurane may possibly reduce prostacyclin formation. Thus, we compared the influences of sevoflurane and isoflurane on endothelial prostacyclin production. Production of prostacyclin of human umbilical vein endothelial cells was measured by the ELISA technique under basal conditions and after stimulation with calcium ionophore A 23187 10 micromol or histamine 0.1 micromol in the absence and presence of 1 and 2 minimal alveolar concentrations (MAC) of sevoflurane or isoflurane. The basal production of prostacyclin was unaffected by the volatile anaesthetics. Stimulation of endothelial cells increased prostacyclin formation 3-5-fold. Sevoflurane at 2 MAC, but not at 1 MAC, could reduce stimulated prostacyclin production by about half (P < 0.05). Isoflurane had no inhibitory effect. Inhibition of cyclo-oxygenase function by acetylsalicylic acid abolished the induced burst of prostacyclin formation completely. Sevoflurane, but not isoflurane, can reduce stimulated endothelial prostacyclin production in a concentration-dependent manner. Because at least 2 MAC of sevoflurane were required, this effect should be of minor importance under clinical conditions of balanced anaesthesia.

Progestogens stimulate prostacyclin production by human endothelial cells

The effects of progestogens on endothelial physiology are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. We examined the effects of two clinically used progestogens, progesterone and medroxyprogesterone acetate (MPA), on prostacyclin production by cultured human umbilical vein endothelial cells (HUVEC) and the possible role of progesterone receptors and both COX enzymes. Cells were exposed to 1-100 nmol/l of either progesterone or MPA and prostacyclin production was measured in culture medium. Both progestogens significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 h. Progesterone and MPA, both at 10 nmol/l, increased mRNA expression and protein content of both COX. All these effects were mediated through progesterone receptor activation, since they were abolished by treatment of cells with the progesterone receptor antagonist RU-486. Selective inhibitors of COX-1 and -2 (SC-560 and NS-398 respectively) reduced basal prostacyclin release, and eliminated increased production in response to progestogens. In combination with estradiol, progestogens had an additive effect without eliminating estradiol-induced prostacyclin production. Our results support the hypothesis that progesterone and MPA increased HUVEC prostacyclin production in a progesterone receptor-dependent manner, by enhancing COX-1 and COX-2 expression and activities.

Vascular Endothelial Growth Factor (VEGF)-D and VEGF-A Differentially Regulate KDR-mediated Signaling and Biological Function in Vascular Endothelial Cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.

Sevoflurane but not isoflurane can reduce prostacyclin production of endothelial cells

Background and objective: Little is known about the interaction of newer volatile anaesthetics with endothelial eicosanoid production. Sevoflurane may possibly reduce prostacyclin formation. Thus, we compared the influences of sevoflurane and isoflurane on endothelial prostacyclin production. Methods: Production of prostacyclin of human umbilical vein endothelial cells was measured by the ELISA technique under basal conditions and after stimulation with calcium ionophore A 23187 10 μmol or histamine 0.1 μmol in the absence and presence of 1 and 2 minimal alveolar concentrations (MAC) of sevoflurane or isoflurane. Results: The basal production of prostacyclin was unaffected by the volatile anaesthetics. Stimulation of endothelial cells increased prostacyclin formation 3-5-fold. Sevoflurane at 2 MAC, but not at 1 MAC, could reduce stimulated prostacyclin production by about half (P < 0.05). Isoflurane had no inhibitory effect. Inhibition of cyclo-oxygenase function by acetylsalicylic acid abolished the induced burst of prostacyclin formation completely. Conclusions: Sevoflurane, but not isoflurane, can reduce stimulated endothelial prostacyclin production in a concentration-dependent manner. Because at least 2 MAC of sevoflurane were required, this effect should be of minor importance under clinical conditions of balanced anaesthesia.

Advanced Glycation End Product-induced Apoptosis and Overexpression of Vascular Endothelial Growth Factor and Monocyte Chemoattractant Protein-1 in Human-cultured Mesangial Cells

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.

Administration of transdermal estrogen without progestin increases the capacity of plasma and serum to stimulate prostacyclin production in human vascular endothelial cells

Objective: To determine whether transdermal hormone replacement therapy modifies the ability of plasma or serum to regulate the synthesis of prostacyclin and that of endothelin-1 by cultured human umbilical vein endothelial cells. Design: Prospective, randomized study. Setting: Department of Obstetrics and Gynecology, Helsinki University Central Hospital. Patient(s): Thirteen postmenopausal women with climacteric symptoms. Interventions: Transdermal 17β-E 2 (50 μg/d) continuously combined with norethisterone acetate, (250 μg/d) on days 15–28 of the treatment cycles for 6 months. Main Outcome Measure(s): Levels of prostacyclin’s metabolite 6-keto-prostaglandin F 1α and of endothelin-1 released by cultured human umbilical vein endothelial cells. Result(s): Plasma and serum during the E 2-only phase of hormone replacement therapy enhanced prostacyclin production by 20% ± 8% (mean ± SEM) and 23% ± 11%, respectively. Plasma or serum taken during the E 2 + norethisterone acetate phase failed to affect prostacyclin production. Hormone replacement therapy induced no change in the capacity of plasma or serum to release endothelin-1. Conclusion(s): Transdermal hormone replacement therapy during the E 2-only phase increased the capacity of plasma and serum to enhance production of vasoprotective prostacyclin in human vascular endothelial cells, without affecting production of endothelin-1. Addition of norethisterone acetate prevented this stimulation.

Effect of estradiol metabolites on prostacyclin synthesis in human endothelial cell cultures

Estradiol can stimulate prostacyclin production in the vessel wall, thereby eliciting vasodilatation. In the present work the effect of the estradiol metabolites estrone, 2-methoxyestrone, 2-methoxyestradiol, and 16α-hydroxyestrone were investigated to find out if they are also able to stimulate prostacyclin synthesis. All metabolites triggered an increase of prostacyclin synthesis in human endothelial cells starting at a concentration of 10 −9 M. The parent substance, 17β-estradiol, accomplished this effect only starting at a concentration of 10 −8 M. These results indicate that estradiol metabolites may take part in the estradiol-induced vasodilatation in vivo.

The role of cyclooxygenase-1 and cyclooxygenase-2 in lipopolysaccharide and interleukin-1 stimulated enterocyte prostanoid formation.

Lipopolysaccharide is an inflammatory agent and interleukin-1 is a cytokine. Their pro-inflammatory effects may be mediated by prostanoids produced by inducible cyclooxygenase-2. The aim of this study was to determine the prostanoids produced by lipopolysaccharide and interleukin-1 stimulated enterocytes through the cyclooxygenase-1 and 2 pathways. Cultured enterocytes were stimulated with lipopolysaccharide or interleukin-1beta with and without cyclooxygenase inhibitors. Low concentrations of indomethacin and valerylsalicylic acid (VSA) were evaluated as cyclooxygenase-1 inhibitors and their effects compared with the effects of a specific cyclooxygenase-2 inhibitor, SC-58125. Prostaglandin E2, 6-keto prostaglandin F1alpha, prostaglandin D2 and leukotriene B4 levels were determined by radioimmunoassay. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme (COX-2) was expressed by 4 h in LPS and IL-1beta treated cells while the constitutive COX-1 remained unaltered in its expression. Interleukin-1beta and lipopolysaccharide stimulated the formation of all prostanoids compared with untreated cells, but failed to stimulate leukotriene B4. Indomethacin at 20 microM concentration, and VSA inhibited lipopolysaccharide and interleukin 1beta stimulated prostaglandin E2, but not 6-keto prostaglandin F1alpha formation. SC-58125 inhibited lipopolysaccharide and interleukin-1beta stimulated 6-keto prostaglandin F1alpha but not prostaglandin E2 release. The specific cyclooxygenase-2 inhibitor also inhibited lipopolysaccharide produced prostaglandin D2 but not interleukin-1beta stimulated prostaglandin D2. While SC-58125 inhibited basal 6-keto prostaglandin-F1alpha formation it significantly increased basal prostaglandin E2 and prostaglandin D2 formation. As SC-58125 inhibited lipopolysaccharide and interleukin-1beta induced 6-keto prostaglandin F1alpha production but not prostaglandin E2 production, it suggests that these agents stimulate prostacyclin production through a cyclooxygenase-2 mediated mechanism and prostaglandin E2 production occurs through a cyclooxygenase-1 mediated mechanism. Prostaglandin D2 production appeared to be variably produced by cyclooxygenase-1 or cyclooxygenase-2, depending on the stimulus.

Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A 2 in endothelial cells via p42/p44 mitogen-activated protein kinase

Vascular endothelial growth factor (VEGF) stimulated a time- and concentration-dependent increase in PGI 2 synthesis in human umbilical vein endothelial cells with a mean maximum increase of 2-fold above basal levels at 25 ng/ml after 60 min. VEGF also rapidly stimulated the release of arachidonic acid and phosphorylation and activation of cytosolic phospholipase A 2 (cPLA 2). The VEGF-related factor, placenta growth factor (PlGF), had little effect on PGI 2 synthesis, arachidonic acid release or cPLA 2 activation. PD98059, a selective inhibitor of MAP kinase kinase, caused complete inhibition of VEGF-stimulated MAP kinase activity, PGI 2 synthesis and cPLA 2 gel retardation, but had no effect on VEGF-induced vWF secretion. These findings provide the first evidence that VEGF can stimulate PGI 2 synthesis via cPLA 2-mediated arachidonic acid release and indicate that VEGF stimulation of this biosynthetic pathway may occur, at least in part, via activation of p42/p44 MAP kinases.

Effects of Oxygen Tension and Shear Stress on Human Endothelial Cell Prostacyclin Production

Underin vivoconditions of ischemia and reperfusion, vascular endothelium (EC) experience concurrent changes in oxygen tension, shear stress, and the local concentration of metabolites. These studies explored the combined effects of shear stress and oxygen tension on EC prostacyclin production. EC grown on microcarrier beads were exposed to 120 min of normoxia and basal shear stress by stirring at 20 rpm. After normoxia, EC were exposed to hypoxia (2% 02, 20 rpm), ischemia (2% 02, 5 rpm) or sham ischemia (20% 02, 5 rpm). Following hypoxia, EC were reoxygenated (20% 02, 20 rpm). After ischemia and sham ischemia, EC were reperfused (20% 02, 20 rpm). Minimal accumulation of metabolites occurred during normoxia, hypoxia, and reperfusion. All metabolites were allowed to accumulate in the flasks during ischemia and sham ischemia. Prostacyclin levels were measured by ELISA, and prostaglandin H2synthase levels in cells were analyzed by immunoblotting. An acute decrease in shear stress decreased prostacyclin production. An acute decrease only in oxygen tension did not alter prostacyclin production significantly. The combined acute decrease in both shear stress and oxygen tension significantly stimulated prostacyclin production for 30 min. By 120 min of ischemia and hypoxia, prostacyclin release was significantly less than sham ischemia. Prostacyclin production after 30 min of reoxygenation was significantly less than that of cells subjected to reperfusion. By 120 min of reperfusion and reoxygenation, there was no significant difference in EC prostacyclin synthesis. These findings suggest that temporal and quantitative aspects of EC prostaglandin synthesis are dependent on both oxygen tension and shear stress.

Hormone replacement therapy modifies the capacity of plasma and serum to regulate prostacyclin and endothelin-1 production in human vascular endothelial cells

To determine if hormone replacement therapy (HRT) modifies the ability of plasma or serum to regulate the synthesis of vasodilatory prostacyclin and that of vasoconstrictive endothelin-1 by cultured human umbilical vein endothelial cells. Plasma and serum collected before and during the sixth treatment cycle of HRT from 13 healthy postmenopausal women were added to cultured endothelial cells. Helsinki University Central Hospital, Department of Obstetrics and Gynecology, Helsinki, Finland. Thirteen postmenopausal women (> or = 1 year since their last menstruation, FSH level > 40 mIU/mL [conversion factor to SI unit, 1.00], clear vasomotor symptoms) that suffered from incapacitating menopausal symptoms necessitating the initiation of HRT were studied. A combined regimen consisting of 2 mg oral E2 for 12 days followed by 2.0 mg oral E2 + 1.0 mg norethisterone acetate for 10 days and 1.0 mg E2 for 6 days. The releases of prostacyclin, as assessed by its metabolite 6-keto-prostaglandin F1 alpha, and that of endothelin-1 by cultured human umbilical vein endothelial cells in the presence of 10% plasma or 10% serum collected from the study subjects. Hormone replacement therapy enhanced the ability of plasma to stimulate prostacyclin production by 21% +/- 6% (mean +/- SEM) during the E2 + norethisterone acetate phase and tended to do so also during the E2-only phase (11% +/- 10%) but caused no change in endothelin-1 release. In contrast, HRT decreased the ability of serum to stimulate prostacyclin production by 12% +/- 5% during the E2-only phase and increased that of endothelin-1 by 8% +/- 4% during the E2 + norethisterone acetate phase. Because plasma flushes endothelial cells in vivo, our data on the HRT-induced stimulation of the capacity of plasma to enhance the production of vasoprotective prostacyclin without a concomitant change in endothelin-1 release in cultured human umbilical vein endothelial cells may provide one new explanation for the cardiovascular protection of HRT.

A 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid, is increased in diabetics with incipient and early renal disease.

Earlier studies in diabetic animal models or ex vivo from diabetics suggest a deficiency in prostacyclin (PGI2) production and an increase in an alternate arachidonic acid metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), which stimulates angiogenesis, mitogenesis, and inhibits renin secretion. We studied the urinary excretion rate of 6-keto-PGF1 alpha (a stable metabolite of PGI2) and 12-HETE in controls and 42 noninsulin-dependent diabetes mellitus (NIDDM) patients with normal renal function and those with micro- or macroalbuminuria/hyporeninemic hypoaldosteronism (HH). The 2 eicosanoids were measured in urine using previously described high pressure liquid chromatography and RIA methods. Normal subjects and patients with NIDDM and microalbuminuria were infused with low dose calcium infusions that stimulate prostacyclin production in normal subjects. The PGI2 excretion rate of NIDDM patients with normal renal function was not different from that of controls (143 +/- 17 vs. 118 +/- 34 ng/g creatinine), but was reduced in those with microalbuminuria (75 +/- 10) and in macroalbuminuria patients (48 +/- 7; P < 0.01). In contrast, 12-HETE was increased in diabetics with normal renal function as well as in those with micro- or macroalbuminuria patients (69 +/- 18 vs. 250 +/- 62 vs. 226 +/- 60 and 404 +/- 131 ng/g creatinine; P < 0.01). Calcium did not stimulate PGI2, but increased 12-HETE in diabetics with microalbuminuria in contrast to levels in normal subjects. HH patients excreted less PGI2 (as previously reported), but had increased 12-HETE. HETE/PGI2 ratios further demonstrated these changes in the various groups. In a nondiabetic hypertensive microalbuminuria group, 12-HETE excretion was normal (73 +/- 28 ng/g creatinine). We conclude that the lipoxygenase product 12-HETE is increased early in the diabetic process, whereas PGI2 production is progressively impaired in NIDDM. These changes may play a role in the vascular disease of diabetes and partially explain the HH syndrome.

Studies on the Characterization of the Subtype(S) of Muscarinic Receptor Involved in Prostacyclin Synthesis in Rabbit Cardiomyocytes

The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin (PGI2) production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased PGI2 synthesis measured as 6-keto-PGF1 alpha, in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose dependent manner but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343, a partially selective M1 muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF1 alpha synthesis in these cell types. ACh induced 6-keto-PGF1 alpha synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (10(-8) M) of pirenzipine, an M1 mAChR antagonist but was reduced by a higher concentration (10(-6) M). In coronary endothelial cells ACh induced 6-keto-PGF1 alpha production was reduced by hexahydro-sila-difendial (HHSiD), an M3 mAChR antagonist, and in ventricular myocytes by both 11-¿2-[(di-ethylamino) methyl]-1-piperidinyl]acetyl-5,11-dihydro-6-H-pyrido-[2,3-b]-benzodiazep ine-6 one¿ (AF-DX 116), an M2 receptor antagonist, and HHSiD. The decrease by ACh of isoporterenol stimulated cAMP accumulation was minimized by AF-DX 116 but not by HHSiD or pirenzipine. Pertussis toxin treatment minimized ACh induced decrease in isoproterenol stimulated rise in cAMP and ATP release, but not ACh induced 6-keto-PGF1 alpha synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M3 mAChR and in ventricular myocytes M2 and M3 mAChR. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF1 alpha production, is mediated by pertussis toxin sensitive G alpha i proteins in these cells.