Increased Prostacyclin Production Research Articles

The Effect of the Extra Virgin Olive Oil Minor Phenolic Compound 3',4'-Dihydroxyphenylglycol in Experimental Diabetic Kidney Disease.

The aim of this study was to analyze the possible nephroprotective effect of 3',4'-dihydroxyphenylglycol (DHPG), a polyphenolic compound of extra virgin olive oil (EVOO), on renal lesions in an experimental model of type 1 diabetes. Rats were distributed as follows: healthy normoglycemic rats (NDR), diabetic rats treated with saline (DR), and DR treated with 0.5 mg/kg/day or 1 mg/kg/day of DHPG. DR showed a significantly higher serum and renal oxidative and nitrosative stress profile than NDR, as well as reduced prostacyclin production and renal damage (defined as urinary protein excretion, reduced creatinine clearance, increased glomerular volume, and increased glomerulosclerosis index). DHPG reduced the oxidative and nitrosative stress and increased prostacyclin production (a 59.2% reduction in DR and 34.7-7.8% reduction in DHPG-treated rats), as well as 38-56% reduction in urinary protein excretion and 22-46% reduction in glomerular morphological parameters (after the treatment with 0.5 or 1 mg/kg/day, respectively). Conclusions: DHPG administration to type 1-like diabetic rats exerts a nephroprotective effect probably due to the sum of its antioxidant (Pearson's coefficient 0.68-0.74), antinitrosative (Pearson's coefficient 0.83), and prostacyclin production regulator (Pearson's coefficient 0.75) effects.

Late Onset of Estrogen Therapy Impairs Carotid Function of Senescent Females in Association with Altered Prostanoid Balance and Upregulation of the Variant ERα36.

Recent analysis of clinical trials on estrogen therapy proposes the existence of a therapeutic window of opportunity for the cardiovascular benefits of estrogens, which depend on women’s age and the onset of therapy initiation. In this study, we aimed to determine how vascular senescence and the onset of estrogen treatment influence the common carotid artery (CCA) function in senescent and non-senescent females. Ovariectomized female senescence-accelerated (SAMP8) or non-senescent (SAMR1) mice were treated with vehicle (OVX) or 17β-estradiol starting at the day of ovariectomy (early-onset, E2E) or 45 days after surgery (late-onset, E2L). In SAMR1, both treatments, E2E and E2L, reduced constriction to phenylephrine (Phe) in CCA [(AUC) OVX: 193.8 ± 15.5; E2E: 128.1 ± 11.6; E2L: 130.2 ± 15.8, p = 0.004] in association with positive regulation of NO/O2- ratio and increased prostacyclin production. In contrast, E2E treatment did not modify vasoconstrictor responses to Phe in OVX-SAMP8 and, yet, E2L increased Phe vasoconstriction [(AUC) OVX: 165.3 ± 10; E2E: 183.3 ± 11.1; E2L: 256.3 ± 30.4, p = 0.005]. Increased vasoconstriction in E2L-SAMP8 was associated with augmented thromboxane A2 and reduced NO production. Analysis of wild-type receptor alpha (ERα66) expression and its variants revealed an increased expression of ERα36 in E2L-SAMP8 in correlation with unfavorable effects of estrogen in those animals. In conclusion, estrogen exerts beneficial effects in non-senescent CCA, regardless of the initiation of the therapy. In senescent CCA, however, estrogen loses its beneficial action even when administered shortly after ovariectomy and may become detrimental when given late after ovariectomy. Aging and onset of estrogen treatment are two critical factors in the mechanism of action of this hormone in CCA.

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.

The Effect of Hawthorn Extract on Coronary Flow

Hawthorn extract has been used for heart failure and may decrease cardiac cell injury and improve cardiac function. One proposed mechanism for hawthorn action is vasodilation. We hypothesized that hawthorn extract would increase coronary blood flow in isolated perfused rat hearts. Coronary flow was measured in nonworking perfused rat hearts (Langendorff, constant pressure) using a flow probe; data were collected electronically in real time. Hawthorn extract showed an early (30-120 seconds) vasodilation, followed by a later (3-5 minutes) decrease in coronary flow. Maximum vasodilation occurred with 240 μg/mL hawthorn extract. Hawthorn’s pattern of activity was unlike that of several known vasoactive drugs. Both nitric oxide synthase inhibitors and indomethacin abolished early vasodilation, but they had no effect on the late phase decrease in flow. We suggest that a hawthorn-induced increase in nitric oxide generation leads to an increase in prostacyclin production, thus causing early phase vasodilation.

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.

Abstract 8573: L-carnitine Prevents Heart Failure with Preserved Ejection Fraction in Associated with Attenuation of Cardiac Fibrosis

Backgrounds: Prognosis of heart failure with preserved ejection fraction (HFpEF) has not been improved because of unknown pathophysiology and unestablished therapeutic strategy. Cardiac fibrosis is known to play a crucial role in the development of HFpEF. Methods and Results: The comprehensive and quantitative analysis of metabolites with capillary electrophoresis time-of-flight mass spectrometry was conducted to find a substance which may be related to the development of HFpEF, and revealed decreased plasma free-carnitine levels in Dahl salt-sensitive rats (DSR) fed high-salt diet, a model of hypertensive HFpEF. We confirmed this result with enzymatic cycling method by showing decreased plasma and left ventricular (LV) free-carnitine levels with increased urinary free-carnitine excretion in HFpEF rats compared to control DSR fed normal chow. We also found that blood free-carnitine levels were decreased in HFpEF patients compared to age-matched normal volunteers. Expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was decreased in the LV and kidney of HFpEF rats. L-carnitine administration to HFpEF rats increased plasma and LV levels of free-carnitine, and prevented HFpEF with decreased myocardial stiffness constant and decreased area of LV fibrosis. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced increase in 3 H-proline incorporation. L-carnitine increased prostacyclin production and expression of fatty acid desaturase 1/2, rate-limiting enzymes in forming arachidonic acid, in the LV of DSR and cultured cardiac fibroblasts. LV arachidonic acid levels were increased in HFpEF rats by L-carnitine administration. Conclusions: L-carnitine may be effective in the prevention of HFpEF through enhanced production of prostacyclin and prevention of LV fibrosis and stiffening. L-carnitine supplementation may become a therapeutic option for HFpEF.

Pulmonary Arterial Responses to Reactive Oxygen Species Are Altered in Newborn Piglets With Chronic Hypoxia-Induced Pulmonary Hypertension

Reactive oxygen species (ROS) have been implicated in the pathogenesis of pulmonary hypertension. ROS might mediate vascular responses, at least in part, by stimulating prostanoid production. Our goals were to determine whether the effect of ROS on vascular tone is altered in resistance pulmonary arteries (PRAs) of newborn piglets with chronic hypoxia-induced pulmonary hypertension and the role, if any, of prostanoids in ROS-mediated responses. In cannulated, pressurized PRA, ROS generated by xanthine (X) plus xanthine oxidase (XO) had minimal effect on vascular tone in control piglets but caused significant vasoconstriction in hypoxic piglets. Both cyclooxygenase inhibition with indomethacin and thromboxane synthase inhibition with dazoxiben significantly blunted constriction to X+XO in hypoxic PRA. X+XO increased prostacyclin production (70 ± 8%) by a greater degree than thromboxane production (50 ± 6%) in control PRA; this was not the case in hypoxic PRA where the increases in prostacyclin and thromboxane production were not statistically different (78 ± 13% versus 216 ± 93%, respectively). Thromboxane synthase expression was increased in PRA from hypoxic piglets, whereas prostacyclin synthase expression was similar in PRA from hypoxic and control piglets. Under conditions of chronic hypoxia, altered vascular responses to ROS may contribute to pulmonary hypertension by a mechanism that involves the prostanoid vasoconstrictor, thromboxane.

BCP crystals increase prostacyclin production and upregulate the prostacyclin receptor in OA synovial fibroblasts: potential effects on mPGES1 and MMP-13

To investigate the potential involvement of prostacyclin in basic calcium phosphate (BCP) crystal-induced responses in osteoarthritic synovial fibroblasts (OASF). OASF grown in culture were stimulated with BCP crystals. Prostacyclin production was measured by enzyme immunoassay. Expression of messenger RNA (mRNA) transcripts was assessed by real-time polymerase chain reaction (PCR). Expression of prostacyclin synthase (PGIS) and the prostacyclin (IP) receptor was measured. The effects of iloprost, a prostacyclin analogue, on expression of genes implicated in osteoarthritis such as microsomal prostaglandin E2 synthase 1 (mPGES1) and matrix metalloproteinases (MMPs) were also studied. FPT inhibitor II, a farnesyl transferase inhibitor, was used to antagonize iloprost-induced responses. BCP crystal stimulation led to a five-fold increase in prostacyclin production in OASF compared to untreated cells. This induction was attenuated by cyclooxygenase (COX)-2 and COX-1 inhibition at 4 and 32h, respectively. PGIS and IP receptor transcripts were constitutively expressed in OASF. BCP crystals upregulated IP receptor expression two-fold. While iloprost diminished BCP crystal-stimulated IP receptor upregulation, the inhibitory effect of iloprost was blocked by the farnesyl transferase inhibitor. In addition, iloprost upregulated mPGES1 and downregulated MMP-13 expression in BCP crystal-stimulated OASF, effects that were not influenced by the farnesyl transferase inhibitor. These data showed for the first time that BCP crystals can increase prostacyclin production and upregulate expression of the IP receptor in OASF. The potential of prostacyclin to influence BCP crystal-stimulated responses was supported by the effects of iloprost on the expression of the IP receptor, mPGES1 and MMP-13. These data demonstrate the potential involvement of prostacyclin in BCP crystal-associated osteoarthritis (OA) and suggest that inhibition of PG synthesis with non-steroidal anti-inflammatory drugs may have both deleterious and beneficial effects in BCP crystal-associated OA.

Effects of vitamin E on the toxicity of oxidized LDL on endothelial cells in vitro in smokers vs nonsmokers on diets rich in fish

To clarify whether supplementation of vitamin E can alter the low density lipoprotein (LDL) oxidation properties and thereby affect endothelial cell function and prostacyclin production in smokers compared to nonsmokers on diets rich in fish in a pilot study. The LDL of six smokers and six nonsmokers on habitual high fish diet was isolated before and after an 8-week supplementation of vitamin E (800 IU/day). LDL was oxidized by incubation with CuSO4. Cytotoxicity of LDL oxidized to different degrees on endothelial cells was investigated in vitro in these two groups. Helsinki University Central Hospital; Institute of Biomedicine, Pharmacology, University of Helsinki. At baseline, the rate of oxidation was higher in nonsmokers than in smokers. The lag phase increased significantly after the supplementation of vitamin E both in smokers and nonsmokers. Native LDL dose dependently tended to reduce the viability of endothelial cells in vitro more markedly when isolated from smokers than from nonsmokers. Vitamin E supplementation had no beneficial effect on the cytotoxicity of oxidized LDLs in endothelial cell culture. On the other hand, simultaneous administration of Trolox, the water-soluble analogue of vitamin E, attenuated the LDL cytotoxicity on endothelial cells. The vitamin E supplementation to LDL donors attenuated the increase in prostacyclin production both in smokers and nonsmokers. Supplementation of LDL donors (healthy male volunteers on habitual fish diet) with vitamin E increased the lag phase of LDL oxidation, but, on the other hand, did not influence in vitro cytotoxicity of LDL, or prostacyclin production.

Effects of Phytoestrogens Genistein and Daidzein on Prostacyclin Production by Human Endothelial Cells

The molecular mechanisms of the vascular effects of phytoestrogens are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. This study examine the effects of two phytoestrogens, the isoflavones genistein and daidzein, on prostacyclin production by cultured human umbilical vein endothelial cells (HUVECs) and the possible role of not only estrogen receptors but also both COX isoforms. The two phytoestrogens significantly increased prostacyclin release in a time- and dose-dependent (0.01-1 microM) manner, being higher than control after 24 h. Selective inhibitors of COX-1, SC-560 [5-(4-chlorophenyl)-1-(4-methoxypjenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2, NS-398 (N-[2-(cyclohexyloxy)-4 nitrophenyl]-methanesulfonamide), were used to investigate the relative contribution of each enzyme. Both inhibitors decreased basal production of prostacyclin, but only COX-2 inhibition completely abolished the isoflavone-stimulated prostacyclin production. Phytoestrogens also increased COX-2 mRNA expression and protein content without affecting COX-1 levels. All these effects were mediated through estrogen receptor activation since treatment of cells with the estrogen receptor antagonist ICI 182780 [7alpha-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta diol] completely abolished the isoflavone-induced increase in prostacyclin production, COX-2 mRNA expression, and COX-2 protein content. The results clearly support the hypothesis that genistein and daidzein increased HUVEC prostacyclin production through estrogen receptor-dependent mechanism, which involved the enhancement of COX-2 protein and activity.

Osteoblast-like cells from estrogen receptor alpha knockout mice have deficient responses to mechanical strain.

In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) alpha and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERalpha. We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERalpha knockout (ERKO) mice (ERalpha-/-) with those from their wildtype (ERalpha+/+) and heterozygous (ERalpha+/-) littermates and from ER/beta knockout (BERKO) mice (ERbeta+/+, ERbeta+/-, and ERbeta-/-). Whereas ERalpha+/+, ERalpha+/-, ERbeta+/+, and ERbeta-/- cells proliferate in response to a single 10-minute period of cyclic strain, ERalpha-/- cells do not. Transfection of fully functional, but not mutant, ERalpha rescues the proliferative response to strain in these cells. The strain-related response of ERalpha-/- cells is also deficient in that they show no increased activity of an AP-I driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERalpha in the IGF axis and the ability of ERalpha-/- cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERalpha. These data indicate ERalpha's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERalpha expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.

Early endothelial dysfunction following renal mass reduction in rats.

Endothelial dysfunction has been previously described in severely hypertensive rats with renal mass reduction (RMR) receiving large dietary Na loads. Because hypertension and Na loading reduce endothelium-dependent vasodilation, the effect of renal failure per se is unclear. Responses to acetylcholine in noradrenaline-contracted isolated perfused mesenteric arteries were studied. Vessels were obtained from RMR rats kept on a normal diet, 3 and 10 days after surgery, and the results were compared with those from sham-operated rats (SN). The role of three putative mediators of endothelium-dependent vasodilation was assessed using: L-NAME (10(-4) mol L(-1)); indomethacin (INDO, 10(-5) mol L(-1)); and a mixture of charybdotoxin and apamin (C/A, both 10(-7) mol L(-1)), inhibitors of Ca-activated K-channels to mediate the effects of endothelium-derived hyperpolarizing factor (EDHF). Response to acetylcholine but not that to nitroprusside (endothelium-independent) was decreased in RMR. L-NAME reduced further acetylcholine relaxations in SN but not in RMR. By contrary, INDO decreased acetylcholine vasodilation in RMR but had no effect in SN. C/A had similar effects in the SN and RMR rats. The levels of 6-keto prostaglandin F1alpha were elevated in the urine of the RMR rats and were perfusate from the RMR vessels. Endothelial dysfunction occurs early after RMR, even when systolic blood pressure is only minimally elevated and Na intake is normal. This alteration may be because of decreased availability of nitric oxide, partially compensated by increased prostacyclin production.

Modulation of Prostacyclin and Thromboxane Secretion by Cytotrophoblasts from Normal and Pre-eclamptic Human Pregnancies

We and others have previously observed an imbalance in cytotrophoblast secretion of the vasoactive prostanoids prostacyclin and thromboxane A2 in pre-eclampsia. To examine the effects of potential modulators of this imbalance, cytotrophoblasts isolated from normal and pre-eclamptic pregnancies were incubated in the presence of lipopolysaccharide, the calcium ionophore A23187, tumour necrosis factor α, or interleukin 1β, with or without the cyclo-oxygenase inhibitor, indomethacin. Further incubations included the drugs tranylcypromine, a prostacyclin synthetase inhibitor (0.1, 10μm ), or the thromboxane synthetase inhibitor, pirmagrel (0.001, 1μm ). Results showed that cytotrophoblasts from pre-eclamptic pregnancies had increased thromboxane production and significant stimulation of prostacyclin production by lipopolysaccharide and calcium ionophore. Lipopolysaccharide stimulated thromboxane production in normal cytotrophoblasts, while indomethacin almost completely inhibited production of both prostanoids. Tranylcypromine mildly inhibited prostacyclin production in normal cytotrophoblasts only, whereas pirmagrel strongly inhibited thromboxane production in a dose-related manner, with reciprocal increase in prostacyclin production occurring in cytotrophoblasts from pre-eclamptic pregnancies. This study confirmed that cytotrophoblasts from pre-eclamptic women had increased thromboxane production and showed that pirmagrel, at the relatively low dose of 0.001μm , was able to normalize the imbalance of thromboxane and prostacylin production and may therefore warrant further investigation as a treatment for pre-eclampsia.

Enhanced endothelin activity prevents vasodilation to insulin in insulin resistance.

Although insulin-mediated vasodilation is impaired in insulin resistance, the mechanisms of this are unknown. We investigated factors mediating vasoactive responses to insulin in control and insulin-resistant rats. Responses to insulin in small mesenteric arteries from control and insulin-resistant rats were investigated after blocking endothelin-A receptors, cyclooxygenase, nitric oxide synthase, and potassium channels. In addition, insulin's effect on prostacyclin production in small mesenteric blood vessels was assessed by enzyme immunoassay. Insulin induced a concentration-dependent vasodilation in control arteries that was absent in arteries from insulin-resistant rats. However, in the presence of BQ610, an endothelin-A receptor antagonist, the response to insulin was normalized in insulin-resistant arteries. In control arteries, insulin-induced vasodilation was completely inhibited by indomethacin, meclofenamate, glibenclamide, or potassium chloride. In contrast, neither n-nitro-L-arginine nor the combination of charybdotoxin and apamin altered vasodilation to insulin. In insulin-resistant arteries in the presence of BQ610, vasodilation was also inhibited by indomethacin, glibenclamide, and potassium chloride. Insulin increased prostacyclin production in small mesenteric blood vessels from both groups of rats to a similar degree. Insulin-induced vasodilation in small rat mesenteric arteries is mediated through prostacyclin- and ATP-dependent potassium channels. However, insulin-resistant arteries do not vasodilate to insulin unless endothelin-A receptors are blocked. Thus, impaired relaxation to insulin in insulin-resistant rats is due to enhanced vasoconstriction by endothelin, which offsets a normal vasodilatory response to insulin.

Opposite Effects of Nicotinic Acid and Pyridoxine on Systemic Prostacyclin, Thromboxane and Leukotriene Production in Man

The effects of nicotinic acid (2500 mg orally during 12 hr) and pyridoxine (300 mg orally twice daily for seven days) on the excretion of urinary 2,3-dinor-6-ketoprostaglandin F1alpha, 11-dehydrothromboxane B2 and leukotriene E4, the markers of systemic prostacyclin, thromboxane A2 and cysteinyl leukotriene production, respectively, were investigated in healthy male volunteers (n=6-8). Nicotinic acid increased 11-dehydrothromboxane B2 and leukotriene E4 excretions to 2.6- and 2.0 times the initial values (P<0.05), respectively. In the volunteers treated with pyridoxine, 11-dehydrothromboxane B2 and leukotriene E4 excretions were decreased to 70% (P<0.05) and 65% (P<0.01) of the initial values, respectively, but the excretion of 2,3-dinor-6-ketoprostaglandin F1alpha was increased 1.7 times (P<0.01). The results suggest that nicotinic acid increases thromboxane and leukotriene synthesis which may not be beneficial for patients with cardiovascular diseases or asthma. In contrast, the increase in prostacyclin production and the inhibition in thromboxane and leukotriene synthesis by pyridoxine might be beneficial in disorders where the production of prostacyclin is decreased and the formation of thromboxane and cysteinyl leukotrienes is enhanced.

Modulation of COX-2 Expression by Statins in Human Aortic Smooth Muscle Cells: INVOLVEMENT OF GERANYLGERANYLATED PROTEINS

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Effect of an estrogen/statin combination on biochemical markers of endothelial function in human coronary artery cell cultures.

The combination of an estrogen with a statin for therapy of postmenopausal women is of interest because both substance classes exert beneficial effects on the lipid profile. However, both substance classes also elicit positive direct effects on the vasculature. Therefore in the present in vitro study an estrogen/statin combination was investigated for its effect on biochemical markers of endothelial function. In endothelial cell cultures from human coronary arteries, the effect of estradiol/fluvastatin, alone and in equimolar combinations, were tested at the concentrations 0.01, 0.1, and 1 microM. The vasodilator prostacyclin, the vasoconstrictor endothelin, endothelial nitric oxide synthase (responsible for synthesis of the vasodilator nitric oxide), the procoagulatory factor plasminogen activator inhibitor-1, and the monocyte chemoattractant protein were chosen as markers. The estradiol/fluvastatin combination was able to increase prostacyclin production (25-100%) in an additive manner. The reduction of endothelin synthesis in the range of 21-46% was higher with the combination than with the monosubstances; the reduction, however, was not statistically significant. The expression of endothelial nitric oxide synthase was not significantly increased by the combination compared with the monosubstances, however, a tendency to an additive increase was observed. For the synthesis of plasminogen activator inhibitor-1, no significant changes were seen for either the monosubstances or the combination. The synthesis of monocyte chemoattractant protein-1 was decreased by the combination between 21% and 40%; the decrease, however, was not statistically significant compared with the effect of the monosubstances. The effective estradiol concentrations are higher than can be achieved by replacement therapy; in contrast, fluvastatin was effective at concentrations that can be reached during clinical treatment. These results indicate that an estrogen/statin combination exerts beneficial effects on the vasculature that seem to be superior to the effects of the monosubstances. The changes found for the biochemical markers can improve endothelial function. The presented results should encourage the performance of clinical studies in cardiovascular risk patients with estrogen/statin combinations.

Effect of DT-TX 30, a Combined Thromboxane Synthase Inhibitor and Thromboxane Receptor Antagonist, on Retinal Vascularity in Experimental Diabetes Mellitus

Combined thromboxane synthase inhibitors and thromboxane receptors antagonists have been shown to have a beneficial effect on different models of thrombosis in vivo. We studied the action of one of these compounds (DT-TX 30) compared with dazoxiben (a thromboxane synthase inhibitor) on retinal vascularity in streptozotocin-diabetic rats. Ten nondiabetic animals were treated with isotonic saline, 30 (10 animals per group) were given 0.4, 4, and 8 mg kg −1 per day of DT-TX 30 (p.o.) and 30 (10 animals per group) were given 10, 50, and 100 mg kg −1 per day of dazoxiben (p.o.) over a 90-day study period. DT-TX 30 caused a dose-dependent decrease of platelet aggregation and thromboxane B 2 synthesis. There was an increase of 9, 65, and 166% in the synthesis of prostacyclin after treatment with 0.4, 4, and 8 mg kg −1 per day, respectively. Retinal vascularity increased in 51, 72, and 182% of animals in response to the three doses used. Synthesis of prostacyclin and the degree of retinal vascularity showed a linear correlation r 2=0.6528, p<0.00001 . Dazoxiben, at doses that inhibited thromboxane synthesis as much as DT-TX 30, increased prostacyclin production and retinal vascularity with less potency than DT-TX 30. In conclusion, the antagonism of thromboxane receptors may be an important additional effect to the inhibition of thromboxane synthase in the prevention of ischemic retinal lesions in experimental diabetes.

The effects of vascular endothelial growth factor on endothelial cells: A potential role in preeclampsia

Objectives: Preeclampsia is primarily a disorder of the maternal endothelium. An as yet unidentified circulating factor causes widespread alteration in endothelial function, and levels of vascular endothelial growth factor are elevated in preeclampsia. We hypothesized that vascular endothelial growth factor is involved in the alteration of endothelial function and set out to find further evidence for this contention. Study Design: Bovine microvascular endothelial cells (B-88) were cultured in vitro. These cultured cells were then stimulated with vascular endothelial growth factor and with plasma from women with preeclampsia in the presence and absence of anti–vascular endothelial growth factor antibody. Prostacyclin, nitric oxide, and lactate dehydrogenase levels were measured. Results: Vascular endothelial growth factor induced a significant concentration-dependent increase in prostacyclin production but not nitric oxide production. Cells stimulated with plasma from women with preeclampsia showed increases in production of both prostacyclin and nitric oxide. Vascular endothelial growth factor concentration in plasma was correlated with prostacyclin production by stimulated cells. The increase in prostacyclin production that usually followed the addition of plasma did not occur when anti–vascular endothelial growth factor antibody was present. Conclusions: Vascular endothelial growth factor has the ability to alter endothelial cell function in a manner analogous to that of plasma from women with preeclampsia. (Am J Obstet Gynecol 2000;182:176-83.)

Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells.

We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm(2) using a cone-plate device, and the concentrations of 6-keto-PGF(1alpha) and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.