Potential Effects of Nonadherent on Adherent Human Umbilical Venous Endothelial Cells in Cell Culture.

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.

Effect of Iloprost, a Prostacyclin Analogue, on Myocardial Ischemia-Reperfusion Injury

Myocardial ischemia-reperfusion injury continues to be observed during open heart surgery. Various experimental models have been developed to overcome this injury and to increase postoperative prognosis. This study was conducted to assess the effect that iloprost, a prostacyclin analogue, can have on myocardial ischemia-reperfusion injury. We evaluated tissue damage by measuring the levels of malonyldialdehyde (MDA), glutathione, and nitric oxide (NO) in tissue and perfusates. In this study, 20 guinea pig hearts were prepared by using the modified Langendorff perfusion apparatus to form control (n = 10) and experimental study groups (n = 10). Following a preischemic period of perfusion and an ischemic period of 20 minutes, control hearts were perfused with Krebs­Henseleit solution. In the experimental group, iloprost (0.45 µg/kg per hour) was included in the perfusates for the last 10 minutes of the preischemic phase. Following cardiac stabilization, heart rate (pulse/min), contractility (mm), and aortic pressure (mmHg) values were recorded at the end of preischemia, postischemia, and reperfusion. Perfusate and tissue analyses for glutathione, MDA, and NO levels were made in each group at the end of experiments. Iloprost was found to have protective effects against myocardial ischemia by means of increased myocardial contractility, decreased tissue/perfusate glutathione levels and inhibited rise of tissue/perfusate MDA observed in the iloprost-treated experimental group. Future investigations on myocardial ischemia-reperfusion injury must evaluate iloprost-related mechanisms.

Sustained Neurological Recovery After Stroke in Aged Rats Treated With a Novel Prostacyclin Analog.

BACKGROUND AND PURPOSE: Targeting the prostaglandin I2 prostanoid (IP) receptor to reduce stroke injury has been hindered by the lack of selective drugs. MRE-269 is the active metabolite of selexipag showing a high selectivity toward the IP receptor. Selexipag has been recently approved for clinical use in pulmonary hypertension. We hypothesized that postischemic treatment with MRE-269 provides long-lasting neuroprotection with improved neurological outcomes in a clinically relevant rat stroke model. METHODS: Aged male Sprague-Dawley rats underwent transient middle cerebral artery occlusion and were randomly selected to receive either vehicle or MRE-269 (0.25 mg/kg) intravenously starting at 4.5 hours post ischemia. Accelerating rotarod and adhesive removal tests were conducted before and at 3, 7, 14, and 21 days after stroke. Infarct volume was quantified by magnetic resonance imaging at 48 hours and 21 days post middle cerebral artery occlusion. In parallel experiments, cerebral cortex samples from stroke and nonstroke sides from vehicle- and MRE-269-treated groups were collected at 18 hours post middle cerebral artery occlusion for molecular biology analyses. RESULTS: Quantitative magnetic resonance imaging data showed that postischemic MRE-269 treatment significantly reduced infarct volume compared with vehicle-treated rats at both 48 hours and 3 weeks after stroke. MRE-269 treatment resulted in a significant long-term recovery in both locomotor and somatosensory functions after middle cerebral artery occlusion, which was associated with a reduced weight loss in animals receiving the IP receptor agonist. Postischemic MRE-269 treatment reduced proinflammatory cytokines/chemokines and oxidative stress. Damage to the blood-brain barrier, as assessed by extravasation of immunoglobulin G to the ischemic brain, was significantly reduced by MRE-269, which was associated with a reduction in matrix metalloproteinase-9 activity in the brain of stroked aged rats given the IP agonist at 4.5 hours after ischemia onset. CONCLUSIONS: Our data suggest that targeting the IP receptor with MRE-269 is a novel strategy to reduce cerebral ischemia injury and promote long-term neurological recovery in ischemic stroke.

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice.

BACKGROUND/AIMS: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. METHODS: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. RESULTS: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin αIIbß3 on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. CONCLUSIONS: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.

Prostaglandin expression profile in hypoxic osteoblastic cells.

Conditions such as fracture and unloading have been shown to be associated with tissue and cellular hypoxia in bone. The effects of hypoxia on bone cell physiology and ultimately its impact on bone tissue repair and remodeling are not well understood. In this study, we investigated the role of hypoxia on prostaglandin release from osteoblastic cells cultured in 2% (hypoxia), 5% (potentially cellular normoxia), and 21% (normoxia for standard cell culture conditions) oxygen for up to 24 h. We quantified the effects of reduced oxygen tension on the release of prostaglandin (PG)E(2), PGF(2alpha), PGD(2), and PGI(2). The mechanism by which hypoxia increases PG production was investigated by examining the various regulatory components of the PG biosynthetic pathway. Our data show that PGE(2) levels alone are significantly elevated under hypoxic conditions. Also, we show that cyclooxygenase (COX)-1 and COX-2 play an important role in hypoxia-induced PGE(2) production, possibly via a mechanism involving changes in their respective activity levels under low oxygen conditions. The effect of hypoxia on PGE(2) levels was mimicked by dimethyloxaloglycine, a known activator of the HIF pathway. In addition, we confirmed that HIF-1alpha was stabilized in osteoblastic cells under hypoxia. Taken together these data suggest a role for the HIF pathway in regulation of PGE(2) levels under hypoxic conditions. Previous studies have detected release of prostaglandins from areas of damaged bone, such as a fracture site, and our data may contribute to an understanding of how this release is regulated.

Estrogen receptor-beta mediates cyclooxygenase-2 expression and vascular prostanoid levels in human placental villous endothelial cells.

OBJECTIVE: Regulation of fetoplacental blood flow is likely mediated by factors such as prostanoids. Estrogen and its receptors affect prostanoid biosynthesis. Previously, we demonstrated that villous endothelial cells express estrogen receptor-beta (ESR2), and we sought to determine its role in the mediation of fetoplacental vascular function. STUDY DESIGN: Villous endothelial cells from uncomplicated pregnancies were isolated, cultured, and treated with estrogen. RNA interference, real-time polymerase chain reaction, Western blotting, and enzyme immunoassays were performed. RESULTS: Cyclooxygenase-2 (COX-2) expression levels were not altered consistently by estrogen. RNA interference of ESR2 led to a concomitant decrease in COX-2 messenger RNA (P < .0001) and protein (P < .05) in the presence and absence of estradiol. ESR2 knock-down also led to diminished prostacyclin and thromboxane concentrations in the absence of estradiol (P < .005). CONCLUSION: ESR2 mediates COX-2 expression levels and both prostacyclin and thromboxane concentrations in the basal state, which suggests the possibility of ligand-independent regulation of COX-2 activity and prostaglandin H2 substrate availability. Further investigation regarding ESR2 regulation of prostanoid biosynthesis and its effects on the fetoplacental vasculature is warranted.

Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells.

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.

Ang II and EGF synergistically induce COX-2 expression via CREB in intestinal epithelial cells.

Cyclooxygenase (COX)-2 derived prostaglandins (PGs) play a major role in intestinal inflammation and colorectal carcinogenesis. Because COX-2 is the rate-limiting step in the production of PGs, mechanisms that regulate COX-2 expression control PG production in the cell. Using the non-tumorigenic, rat intestinal epithelial cell, IEC-18, we demonstrate that co-activation of endogenously expressed AT(1) receptor and EGFR resulted in synergistic expression of COX-2 mRNA and protein involving transcriptional and post-transcriptional mechanisms. Ang II and EGF induced transient phosphorylation of ERK, p38(MAPK) and CREB. Co-stimulation with Ang II and EGF prolonged phosphorylation of ERK, p38(MAPK), and CREB. The p38(MAPK) selective inhibitor, SB202190, but not the MEK selective inhibitor, PD98059, or the EGFR kinase inhibitor, AG1478, inhibited Ang II-dependent COX-2 expression and CREB phosphorylation. EGF-dependent COX-2 expression and CREB phosphorylation were inhibited by SB202190, PD98059, and AG1478. Inhibition of CREB expression using two separate RNAi methods blocked COX-2 expression by Ang II and EGF. Expression of a dominant negative CREB mutant inhibited Ang II- and EGF-dependent induction of the COX-2 promoter. Ang II induced luciferase expression in cells transfected with the CRE-luc reporter vector and cells co-transfected with Gal4-luc reporter vector and a Gal4-CREB expression vector. Chromatin immunoprecipitation assays demonstrated CREB binding to the proximal rat COX-2 promoter region containing a CRE cis-acting element. These results indicate that co-stimulation with Ang II and EGF synergistically induced COX-2 expression in these intestinal epithelial cells through p38(MAPK) mediated signaling cascades that converge onto CREB.

Oxygen and the liberation of placental factors responsible for vascular compromise.

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

Heme oxygenase induction by cyanidin-3-O-beta-glucoside in cultured human endothelial cells.

The aim of the present research was to investigate the effect of cyanidin-3-O-beta-glucoside (C3G) on heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS) and dimethylarginine dimethylamino hydrolase-2 (DDAH-2) expression in cultured endothelial cells. Different concentrations (0.00625-250 microM) of C3G were tested in order to investigate possible beneficial and harmful effects of C3G. Our data demonstrated that C3G increased the induction of eNOS and HO-1 in a dose-dependent manner. Higher concentration (62.5-250 microM) also resulted in increase of isoprostane, cGMP and PGE2 levels and in induction of iNOS with consequent oxidative stress. In conclusion, our data evidence that C3G may exert various protective effects against endothelial dysfunction, whereas potentially harmful effects of C3G appear to be limited to concentrations very difficult to be reached in physiological conditions unless there is abundant oral supplementation.

Roles of nitric oxide and prostacyclin in triethyleneglycol dimethacrylate (TEGDMA)-induced vasorelaxation.

OBJECTIVES: Most dental resinous materials contain the diluent monomer triethyleneglycol dimethacrylate (TEGDMA), which has been reported to be bioactive. Previously, it was demonstrated that TEGDMA induces vasorelaxation. The present study examines the mechanism(s) of the TEGDMA-induced vasorelaxation by measuring vascular nitrite and prostacyclin levels. METHODS: Nitrite and prostacyclin levels were assayed in rat aortic tissues in response to TEGDMA. The involvement of guanylyl and adenylyl cyclases in TEGDMA-induced aortic vasorelaxation was determined using the enzyme inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), respectively. RESULTS: TEGDMA enhanced the levels of nitrites in endothelium-intact and that of protacyclin in both endothelium-intact and denuded rat aortas. The increase in nitrites was associated with endothelium-dependent aortic relaxation mediated via the activation of guanylyl cyclase, while the increase in prostacyclin was associated with both endothelium-dependent and independent relaxation linked to adenylyl cyclase stimulation. SIGNIFICANCE: Data from the present investigation can be relevant to dental practice employing materials containing TEGDMA by providing insights into the vasorelaxant effect of the monomer following placement of the materials in the oral cavity. Additional studies that are more relevant to the clinical situation are required to confirm these initial results and further explore their implications.

Prostacyclin as an indicator of preadipocyte transformation: studies in vivo by microdialysis and in vitro.

Nontransformed (Ob1771) and polyoma virus-transformed (Ob17PY) mouse cells from the preadipocyte Ob17 clonal line have been compared in their ability to release prostaglandins in vitro as well as in vivo as assayed by in situ microdialysis. Prostaglandin FE2, prostaglandin-2 alpha and mainly prostacyclin are released in larger amounts (4- to 10-fold) by Ob17PY cells in vitro and Ob17PY-induced tumors in vivo as compared to Ob1771 preadipocytes in vitro and periepididymal adipose tissue in vivo. In contrast to Ob1771 preadipocytes, none of these prostanoids appear to be involved in the control of proliferation or differentiation of Ob17PY cells in serum-free culture medium. However, prostacyclin, the level of which is the most affected by transformation, might be considered as a valuable indicator of fibrosarcoma development.

Cooperation of adipocytes and endothelial cells required for catecholamine stimulation of PGI2 production by rat adipose tissue.

Disturbances of prostaglandin I2 (PGI2, prostacyclin) production by adipose tissue contribute to the pathogenesis of diabetic ketoacidosis and may contribute to the pathogenesis of hypertension and vascular disease. We studied the cellular basis of PGI2 production in adipose tissue, measured as release of 6-keto-PGF1 alpha in response to epinephrine. Adipocytes did not produce PGI2 when nonfat cells were removed by repeated washing. The nonadipocyte cellular constituents of adipose tissue (nonfat cells) did not produce PGI2 in the absence of adipocytes. Both adipocytes and nonfat cells were required for PGI2 production in response to epinephrine. Adipocytes pretreated with 0.2 mM aspirin to inhibit PGH synthase nevertheless promoted PGI2 production when mixed with nonfat cells. Nonfat cells preincubated with aspirin did not produce PGI2 when mixed with adipocytes. The nonfat cells converted arachidonic acid to PGI2 but adipocytes did not. Epinephrine stimulated lipolysis and PGI2 production in a dose-dependent parallel manner, but the responses were distinct above 10(-6) M. Characterization of the nonfat cells by fractionation on a Percoll density gradient followed by measurement of angiotensin-converting enzyme activity and 6-keto-PGF1 alpha production indicated that the nonfat cells were predominantly vascular endothelial cells. We conclude that catecholamine-stimulated PGI2 production in adipose tissue results from the cooperation of adipocytes and vascular endothelial cells. The adipocytes provide arachidonic acid, which is converted to PGI2 by the vascular endothelial cells. Because adipose tissue is located near blood vessels throughout the body, adipocytes may be an important source of arachidonic acid for vascular endothelial cells in various circumstances in health and disease. Our findings raise the possibility that adipocytes may, under some circumstances, release arachidonic acid into the systemic circulation where it is used by vascular endothelial cells throughout the body to produce PGI2 and other eicosanoids.

Effect of fluid flow on smooth muscle cells in a 3-dimensional collagen gel model.

A 3D collagen gel model was developed to simulate interstitial fluid flow and to assess the importance of this flow on the biochemical production rates of vascular smooth muscle cells (SMCs). Rat aortic SMCs were suspended in type I collagen, and the gel was supported by nylon fibers that allowed a 9-cm length of the SMC-gel model to withstand 90 cm H(2)O differential pressure over a 6-hour period without significant compaction. Up to 1 dyne/cm(2) shear stress on the suspended SMCs could be induced by the pressure-driven interstitial flow. The suspended SMCs were globular, had a diameter of approximately 10 microm, and were distributed uniformly throughout the gel. The collagen fibers formed a network that was connected randomly with the surface of SMCs and nylon fibers. The diameter of the collagen fibers was approximately 100 nm, and the concentration of collagen was 2.5 mg/mL. Using these parameters, fiber matrix theory predicted a Darcy permeability coefficient (K:(p)) of 1.22x10(-)(8) cm(2), which was close to the measured value of K:(p). The production rates of prostaglandin (PG) I(2) and PGE(2) were used as markers of biochemical responsiveness of SMCs to fluid shear stress. Both PGI(2) and PGE(2) production rates under 1 dyne/cm(2) shear stress were significantly elevated relative to static (no-flow) controls. The production rates, however, were approximately 10 times lower than observed when the same cells were plated on collagen-treated glass slides (2D model) and exposed to the same level of shear stress by use of a rotating disk apparatus. The results indicate that interstitial flow can affect SMC biology and that SMCs are more quiescent in 3D cultures than in 2D cultures. The 3D collagen gel model should be useful for future studies of interstitial flow effects on SMC function.

Analysis of fatty acid samples by hydrophilic interaction liquid chromatography and charged aerosol detector.

HILIC/CAD techniques were used in analysis of samples containing fatty acids. Amine base column appeared to be the more retentive stationary phase compared to zwitterionic and BEH silica. The retention decreased with pH mobile phases changing from 3 to 5. Acetonitrile and acetone organic modifier were compared. Acetone gave higher eluotropic strength and better peak symmetry whereas acetonitrile led to higher efficiency. The retention decreased when ammonium acetate concentration increased from 5 to 20mM. The use of sub-2µm column did not show flat Van Deemter curves at high flow rates. A rapid separation of PGI2 and its main degradation product, 6-keto prostaglandin F1α was obtained in 1.6min with a Hypersil GOLD, 50mm×2.1mm, 1.9µm with; acetonitrile/acetate ammonium pH 5 at 20mM (85/15; v/v at 0.7ml/min).

Increased concentrations of arachidonic acid, prostaglandins E2, D2, and 6-oxo-F1 alpha, and histamine in human skin following UVA irradiation.

The buttock skin of clinically normal human subjects was subjected to approximately 2.5 minimal erythema doses of ultraviolet A irradiation. Deep red erythema developed during irradiation, faded slightly within the next few hours, increased to maximum intensity between 9-15 h, and decreased gradually thereafter although still persisting strongly at 48 h. Suction blister exudates were obtained at 0, 5, 9, 15, 24, and 48 h after irradiation as well as suction blister exudates from a contralateral control site and assayed for arachidonic acid, prostaglandins D2 and E2, and the prostacyclin breakdown product 6-oxo-prostaglandin F1 alpha by gas chromatography-mass spectrometry, and for histamine by radioenzyme assay. Increased concentrations of arachidonic acid and prostaglandins D2, E2, and 6-oxo-prostaglandin F1 alpha were found maximally between 5-9 h after irradiation, preceding the phase of maximal erythema. Elevations of histamine concentration occurred 9-15 h after irradiation, preceding and coinciding with the phase of maximal erythema. At 24 h, still at the height of the erythemal response, all values had returned to near control levels. Hence increased concentrations of arachidonic acid and its products from the cyclooxygenase pathway, and of histamine, accompany the early stages up to 24 h. A causal role in production of the erythema seems likely for these substances although other mediators are almost certainly involved.

Cyclooxygenase 2 pathway mediates IL-1beta regulation of IL-1alpha, -1beta, and IL-6 mRNA levels in Leydig cell progenitors.

Prostanoids are arachidonic acid (AA) metabolites derived from the cyclooxygenase (COX1 and COX2 isozymes) pathway and are involved in signal transduction pathways activated by distinct ILs. Although COX1 is the constitutive isoform of COX, IL-1beta is a potent inducer of COX2 expression in distinct cell types. This study was designed to determine whether cyclooxygenases could mediate endogenous cytokine regulation in rat progenitor Leydig cells. COX and IL (IL-1alpha, IL-1beta, and IL-6) mRNAs were measured by PCR and real-time PCR analyses, respectively. COX function was assessed using COX activity inhibitors: indomethacin (INDO; COX1 and COX2 inhibitor) and NS-398 (COX2 selective inhibitor). Our data indicate that endogenous progenitor COX1 mRNA levels are low and are not regulated by IL-1beta. In contrast, COX2 mRNA is induced by IL-1beta at 6, 9, and 24 h. IL-1beta induction of IL mRNAs was in part significantly impaired in the presence of INDO or NS-398. Among the prostanoids tested, prostaglandin E(2) (PGE(2)), PGF(2alpha), and carbaprostacyclin reversed the INDO inhibition of IL production. PGs alone have no (IL-1alpha and IL-1beta) or a modest (IL-6) effect on IL mRNA levels. PGE(2), PGF(2alpha), and PGI(2) measurements show that IL-1beta treatment significantly increases progenitor Leydig cell production of these PGs. Taken together, our data demonstrate that this COX2 cascade is a regulator of cytokines in Leydig progenitors.

Mediators of arachidonic acid-induced relaxation of bovine coronary artery.

Bovine coronary arteries relax in response to bradykinin, methacholine, sodium nitroprusside, isoproterenol, and arachidonic acid in a concentration-dependent manner. The relaxations to methacholine, bradykinin, and arachidonic acid are lost when endothelium is removed. Indomethacin, a cyclooxygenase inhibitor, attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus indomethacin) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus 2 x 10(-9) mo1/L, and 3 x 10(-7) versus 2 x 10(-6) mo1/L, respectively). Nitro-L-arginine, a nitric oxide synthase inhibitor, also attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus nitro-L-arginine) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). The combination of indomethacin and nitro-L-arginine blunted the relaxations to these agents and also shifted the EC50 values (control versus indomethacin plus nitro-L-arginine) to each (1 x 10(-7) versus 5 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). Methacholine, bradykinin, and arachidonic acid stimulated the release of prostaglandin I2, measured as 6-keto-PGF1 alpha. Indomethacin, but not nitro-L-arginine, inhibited arachidonic acid-induced release of 6-keto-PGF1 alpha. Vascular cGMP content was unchanged by arachidonic acid but was significantly elevated by bradykinin. Relaxations to prostaglandin I2 and sodium nitroprusside, but not 8,9-epoxyeicosatrienoic acid or isoproterenol, were inhibited by nitro-L-arginine. We conclude that the endothelium-dependent relaxations to methacholine, bradykinin, and arachidonic acid are partly due to prostaglandin I2 release. The remainder of the responses to these agents is due to the release of other relaxing factor or factors. Since bradykinin increased cGMP and nitro-L-arginine partially inhibited its relaxant effects, nitric oxide also appears to participate in the bradykinin-induced effect. Since the combination of indomethacin and nitro-L-arginine failed to completely block the relaxations to methacholine, bradykinin, and arachidonic acid, another endothelial factor must contribute to their vascular effects. Surprisingly, nitro-L-arginine attenuated the relaxations to arachidonic acid; however, L-arginine failed to reverse the effects of nitro-L-arginine on arachidonic acid-induced relaxations. In addition, arachidonic acid failed to increase cGMP. Nitro-L-arginine also reduced the responses to prostaglandin I2 and sodium nitroprusside. These data indicate that these arginine analogues may have effects other than competitive inhibition of nitric oxide synthase.

Bradykinin-induced reductions in collagen gene expression involve prostacyclin.

Cardiac fibrosis after myocardial infarction and in chronic hypertension involves an increase in the synthesis and deposition of collagen within the myocardium. Angiotensin-converting enzyme (ACE) inhibitors limit hypertrophy and fibrosis; their mechanism of action remains controversial, although kinins have been implicated to play a role. Because both bradykinin and prostaglandins (PG) have been shown to reduce collagen gene expression in cardiac fibroblasts, the goal of this study was to determine whether the bradykinin effect was mediated through enhanced prostaglandin formation by cardiac fibroblasts. Bradykinin increased [3H]arachidonic acid metabolite release 2.3-fold over control and stimulated a dose-dependent increase in 6-keto PGF1alpha (the stable metabolite of PGI2) release from these cells, in which 1 nmol/L bradykinin produced a 4-fold increase in 6-keto PGF1alpha release. Beraprost (a PGI2 analogue) reduced steady-state proalpha1(I) and proalpha1(III) collagen mRNA levels by 35.6+/-6.6% and 34.2+/-10.0%, respectively. Bradykinin-induced reductions in collagen type I and III gene expression were reversed by pretreatment with indomethacin. Our results indicate that one mechanism by which bradykinin modulates collagen biosynthesis via the rabbit cardiac fibroblast involves formation of arachidonic acid metabolites, particularly PGI2. The results of the present study argue that stabilization of endogenous kinins (as by ACE inhibitors) would enhance prostacyclin production and result in the attenuation of collagen gene expression, with potential implications for collagen synthesis and deposition within the myocardium.