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.

Synthesis of prostacyclin and its effect on the contractile activity of the inflamed porcine uterus.

The goal of the study was to estimate the content of prostacyclin (PGI(2)), the levels of PGI synthase (PTGIS) and receptor (PTGIR) protein expression, and the cellular localization of these factors in the inflammatory-changed porcine uterus. The effect of PGI(2) on the contractility of the inflamed uteri was also determined. On Day 3 of the estrous cycle (Day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) colony-forming units/mL) were injected into each uterine horn. Acute endometritis developed in all bacteria-inoculated gilts, however on Day 8 of the study a severe form of acute endometritis was noted more often than on Day 16. Bacteria injections increased the contents of 6-keto-prostaglandin F(1α) in endometrium, myometrium, washings, and the level of PTGIS in endometrium on Days 8 and 16, and the content of PTGIR in endometrium on Day 16. In the inflamed uteri on both study days, stronger immunoreactivity for PTGIS was observed in part of the luminal and glandular epithelial cells and in a portion of the endometrial arteries, and for PTGIR in part of the luminal epithelium and endothelial cells in a portion of the endometrial arteries. On Day 8, PGI(2) decreased contraction intensity in endometrium/myometrium and myometrium of the saline-treated uteri and increased the contraction intensity in both types of strips from the inflamed organs. Our study reveals that inflammation of the porcine uterus upregulates PGI(2) synthesis and that PGI(2) increases contractility, which suggests that PGI(2) might be essential for the course of uterine inflammation.

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.

Concentration of haemodynamic and inflammatory related cytokines in diabetic retinopathy.

AIM: There are changes in blood flow during the clinical stages of diabetic retinopathy with increasing leukostasis and secondary elaboration of cytokines. This study evaluated the vitreous concentrations of haemodynamic-related (endothelin-1 (ET-1) and nitric oxide (NO)), inflammatory and anti-inflammatory (interleukin-1 receptor antagonist, IL-1 Ra) cytokines in the diabetic patients (with nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR)), compared them with those of control patients (full thickness macular hole, FTMH) and correlated to macular structural indices. METHOD: Vitreous samples from five FTMH patients representing normal controls were analysed together with the vitreous samples of 15 patients with NPDR and five with PDR. The vitreous concentrations of nitrite (total NO), ET-1, and prostacyclin was determined using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. A sandwich luminescent immunoassay technique was used to determine IL-1beta and IL-1 Ra concentrations. RESULTS: In the different clinical groups, there were no differences in the vitreous NO and prostacyclin concentrations. In NPDR, the median ET-1 concentration (0.7 pg/ml SD +/-0.8 pg/ml) was significantly reduced (P<0.05), compared to PDR (6.35 pg/ml SD +/-0.6 pg/ml) and FTMH (3.6 pg/ml SD +/-0.14 pg/ml). Its concentration also positively correlated with foveal thickness and macular volume (P<0.05) in patients with NPDR and macular oedema. IL-1 beta was detected in PDR, and diabetic patients demonstrated a lower concentration of the anti-inflammatory cytokine IL-1 Ra. CONCLUSION: Reduced concentrations of ET-1 in NPDR may reflect the haemodynamic changes of NPDR. The IL-1 Ra concentration suggests a change in the anti-inflammatory environment of the diabetic retina.

[Study on the correlation between hypertension and the indexes of vascular endothelial function among people living in the community].

OBJECTIVE: To study the features of hypertension and vessel endothelium functional parameter in people living at the community level as well as the risk factors of hypertension. Differences of angiotensin II (Ang II ), prostacyclin (PGI2) and nitric oxide (NO) among normal group and three hypertension groups were also studied. METHODS: By cluster sampling, 1134 adult Han people were selected from the residential communities. Medical history was documented and measurements of body height, body weight, waist circumference, hip circumference and blood pressure were performed. Serum NO levels were determined by cadmium reduction method while plasma Ang II and PGI2 concentration were determined by radioimmunoassay. SPSS 13.0 was used for data analysis. RESULTS: The total ratio of hypertension from people living at the community was 44.5%, with the standardized prevalence of hypertension as 15.3%. With the increase of age, the prevalence of hypertension also increased. Overweight and obesity seemed to be independent risk factors for hypertension. History of smoking and drinking and gender did not enter the logistic equation for hypertension. The amount of plasma Ang II concentration of the three hypertension groups was significantly lower than that in the normal group while the lowest group appeared to from the one that hypertension was under control. The NO and PGI2 levels of the two groups whose hypertension had been known were significantly higher than in the normal group while the difference between the group whose hypertension had not been measured and the normal group was not found. CONCLUSION: The prevalence of hypertension had been increasing. Control of body weight seemed to be a useful way for prevention of hypertension. We assumed that the negative feedback regulation of renin-angiotonin-aldosterone system in hypertension patient still existed which called for the research on the mechanism of hypertension.

Phytoestrogens increase the capacity of serum to stimulate prostacyclin release in human endothelial cells.

BACKGROUND: Both the estrogen receptor (ER) alpha and beta isoforms are expressed in the endothelium. The ER beta has been assigned a crucial role in normal vascular wall function. Prostacyclin has been ascribed a beneficial effect on vessel wall physiology. Isoflavones bind with higher affinity to ER beta. We investigated the hypothesis that their administration to postmenopausal women can promote endothelial prostacyclin production. METHODS: Twenty-five healthy postmenopausal women with mild climacteric symptoms received capsules containing 55 mg/day isoflavones derived from soy and red clover for 6 months. Cultured human umbilical vein endothelial cells (HUVECs) were exposed for 24 h to serum collected before the initiation of therapy and then after 3 and 6 months of continuous therapy. Prostaglandin production was measured in culture medium. RESULTS: In the presence of serum obtained after isoflavone treatment, the prostacyclin production increased significantly from 2.7 +/- 0.5 ng/mg protein at baseline to 3.4 +/- 0.7 ng/mg protein at 3 months (p < or = 0.05), and to 3.8 +/- 0.7 ng/mg protein at 6 months (p < or = 0.05 vs. baseline and 3 months' treatment). CONCLUSIONS: Serum obtained from postmenopausal women treated with isoflavones stimulates the capacity to produce prostacyclin by HUVECs in culture, an effect that could contribute to a beneficial cardiovascular effect of phytoestrogens.

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.

Effect of endothelin-1 on intracellular glutathione and lipid peroxide availability and on the secretion of vasoactive substances by human umbilical vein endothelial cells.

BACKGROUND: The major pathophysiologic changes observed in preeclampsia suggest that endothelial cell dysfunction plays an important role in this disorder. The pathway mediating endothelial cell layer dysfunction is unknown. The concentration of endothelin-1 (ET-1), a potent mammalian vasoconstrictor peptide produced by the vascular endothelium, has been observed to be significantly increased in preeclampsia. In this study, we determined the in vitro effect of endothelin-1 on glutathione and lipid peroxide levels and on the secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs). METHODS: Human umbilical vein endothelial cells were incubated for 24 h in the presence of different concentrations of ET-1 (0-1000 pmol L(-1)), which were shown in an earlier experiment to have no effects on vitality and proliferation rate of HUVECs. The levels of glutathione (GSH) and lipid peroxides (LPO) were measured in endothelial cell lysates. For the measurement of vasoactive substances, levels of nitric oxide (NO), prostacyclin (PGI2) and thromboxane A2 (TXA2) were measured in endothelial cell supernatants. RESULTS: At lower concentrations (5-50 pmol L(-1)), ET-1 increases the intracellular content of LPO, stimulates the secretion of TXA2, but inhibits the secretion of PGI2 in endothelial cells compared with control cells. At higher concentrations (100-1000 pmol L(-1)), ET-1 increases the intracellular content of GSH, but results in a decrease of LPO, and increase of PGI2, back to control levels. ET-1 has no effect on NO secretion. CONCLUSION: These findings demonstrate that at concentrations corresponding to values in plasma from preeclamptic women, ET-1 induces oxidative stress and results in altered secretion of vasoactive substances in human endothelial cells. We conclude that ET-1 may participate in the pathway leading to endothelial cell dysfunction seen in preeclampsia.

Induction of cyclooxygenase-2 and enhanced release of prostaglandin E(2) and I(2) in human endothelial cells by engagement of CD40.

OBJECTIVES: The hypothesis was tested that CD40-CD154 interaction is involved in the induction of cyclooxygenase-2 and the release of prostanoids in human endothelial cells. METHODS AND RESULTS: In a coculture model of human endothelial cells and a transfected CD154 positive cell line, engagement of CD40 on endothelial cells dramatically increased the synthesis of prostacyclin, prostaglandin E(2) and thromboxane A(2). This upregulation was mediated through an induction of cyclooxygenase-2 (Cox-2), as it was blocked by Cox-2-selective inhibitors. Western blot analysis demonstrated that Cox-2 protein was markedly increased in endothelial cells following CD40 engagement, an effect that was inhibited by pretreatment of cells with an anti-CD154 antibody. CONCLUSION: The data indicate that signaling via CD40 constitutes a major pathway in human endothelial cells for the induction of Cox-2 and release of prostanoids. The CD40-Cox-2 axis thus may represent an important pathway for initiating or maintaining an inflammatory process at the vessel wall.

Prostacyclin and hormone levels in patients with symptoms of miscarriage and infection.

During pregnancy, acsending infection into the uterus, is followed by local increase of cyclooxygenase-2 (COX-2) activity, and consequently elevated prostaglandin production. Subclinical infections of fetal amniotic membranes are well known to cause preterm delivery. Spreading of infection from vagina and cervix into uterine cavity, can be tragic for the fetus. The aim of this study was to reveal wether lower genital tract infections are associated with symptoms of threatened miscarriage. Our investigation included 140 patients with symptoms of threatened miscarriage and 70 patients with uncomplicated pregnancies. Infections were detected by vaginal or cervical smears as well as tests for Chlamydial infection. Levels of prostacyclin, main prostaglandin product during pregnancy, were evaluated via its stable metabololite, 6-keto-PGF1-alpha. Both prostaglandin levels and hormones were determined by ELISA method. We found that serum values of prostacyclin were elevated, while levels of estradiol, progesterone and prolactin were significantly lower in patients with lower genital tract infections. Increased prostacyclin levels in pregnancies complicated by lower genital tract infections are caused by stimulation of COX/2 enzyme, due to elevated production of various cytokines which are possibly a compensatory mechanism resolving problems caused by bacterial endotoxines.

Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer.

BACKGROUND: We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids. METHODS AND RESULTS: ECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2alpha were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis. CONCLUSIONS: Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

Viral IL-10 gene transfer decreases inflammation and cell adhesion molecule expression in a rat model of venous thrombosis.

Post-thrombotic inflammation probably contributes to chronic venous insufficiency, and little effective treatment exists. IL-10 is an anti-inflammatory cytokine that previously has been shown to decrease perithrombotic inflammation and thrombosis. We investigated in a rat model whether local expression of viral IL-10 (vIL-10) in a segment of vein that undergoes thrombosis would confer an anti-inflammatory effect and how this effect might be mediated. Rats underwent inferior vena cava isolation, cannulation, and instillation of saline or adenovirus encoding either beta-galactosidase or vIL-10. Two days after transfection, thrombosis was induced, 2 days after this the rats underwent gadolinium (Gd)-enhanced magnetic resonance venography exam, and the vein segments were harvested. Tissue transfection was confirmed by either RT-PCR of vIL-10 or positive 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-Gal) staining. vIL-10 significantly decreased both leukocyte vein wall extravasation and area of Gd enhancement compared with those in controls, suggesting decreased inflammation. Immunohistochemistry demonstrated decreased endothelial border staining of P- and E-selectin, while ELISA of vein tissue homogenates revealed significantly decreased P- and E-selectin and ICAM-1 levels in the vIL-10 group compared with those in controls. Importantly, native cellular IL-10 was not significantly different between the groups. However, neither clot weight nor coagulation indexes, including tissue factor activity, tissue factor Ag, or von Willebrand factor levels, were significantly affected by local vIL-10 expression. These data suggest that local transfection of vIL-10 decreases venous thrombosis-associated inflammation and cell adhesion molecule expression, but does not directly affect local procoagulant activity.

Inhaled nitric oxide and nifedipine have similar effects on lung cGMP levels in rats.

UNLABELLED: Inhaled nitric oxide (NO) may downregulate the endogenous NO/cyclic guanosine monophosphate (cGMP) pathway, potentially explaining clinical rebound pulmonary hypertension. We determined if inhaled NO decreases pulmonary cGMP levels, if the possible down-regulation is the same as with nifedipine, and if regulation also occurs with the cyclic adenosine monophosphate (cAMP) pathway. Rats were exposed to 3 wk of normoxia, hypoxia (10% O2), or monocrotaline (MCT; single dose = 60 mg/kg) and treated with either nothing (control), inhaled NO (20 ppm), or nifedipine (10 mg x kg(-1) x day(-1). The lungs were then isolated and perfused with physiologic saline. Perfusate cGMP, prostacyclin, and cAMP levels were measured. Perfusate cGMP was not altered by inhaled NO or nifedipine in normoxic or MCT rats. Although hypoxia significantly increased cGMP by 128%, both inhaled NO and nifedipine equally prevented the hypoxic increase. Inhibition of the NO/cGMP pathway with N(G)-nitro-L-arginine methyl ester (L-NAME) decreased cGMP by 72% and 88% in normoxic and hypoxic lungs. Prostacyclin and cAMP levels were not altered by inhaled NO or nifedipine. L-NAME significantly decreased cGMP levels, whereas inhaled NO had no effect on cGMP in normoxic or MCT lungs, suggesting that inhaled NO does not inhibit the NO/cGMP pathway. Inhaled NO decreased cGMP in hypoxic lungs, however, nifedipine had the same effect, which indicates the decrease is not specific to inhaled NO. IMPLICATIONS: High pulmonary pressure after discontinuation of inhaled nitric oxide (NO) may be secondary to a decrease in the natural endogenous NO vasodilator. This rat study suggests that inhaled NO either does not alter endogenous NO or that it has similar effects as nifedipine.

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts.

PURPOSE: To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS: This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS: There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS: (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.

Hyperventilation induces release of cytokines from perfused mouse lung.

Artificial mechanical ventilation represents a major cause of iatrogenic lung damage in intensive care. It is largely unknown which mediators, if any, contribute to the onset of such complications. We investigated whether stress caused by artificial mechanical ventilation leads to induction, synthesis, and release of cytokines or eicosanoids from lung tissue. We used the isolated perfused and ventilated mouse lung where frequent perfusate sampling allows determination of mediator release into the perfusate. Hyperventilation was executed with either negative (NPV) or positive pressure ventilation (PPV) at a transpulmonary pressure that was increased 2.5-fold above normal. Both modes of hyperventilation resulted in an approximately 1.75-fold increased expression of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) mRNA, but not of cyclooxygenase-2 mRNA. After switching to hyperventilation, prostacyclin release into the perfusate increased almost instantaneously from 19 +/- 17 pg/min to 230 +/- 160 pg/min (PPV) or 115 +/- 87 pg/min (NPV). The enhancement in TNFalpha and IL-6 production developed more slowly. In control lungs after 150 min of perfusion and ventilation, TNFalpha and IL-6 production was 23 +/- 20 pg/min and 330 +/- 210 pg/min, respectively. In lungs hyperventilated for 150 min, TNFalpha and IL-6 production were increased to 287 +/- 180 pg/min and more than 1,000 pg/min, respectively. We conclude that artificial ventilation might cause pulmonary and systemic adverse reactions by inducing the release of mediators into the circulation.

Evaluation of a polyester collagen-coated heparin bonded vascular graft.

This animal study was designed to compare a collagen coated heparin bonded vascular graft (CHG) versus a collagen coated vascular graft (CG) regarding intraoperative blood loss and healing process. 24 polyester vascular grafts (12 CHG and 12 CG) of 6 mm in diameter and 5 mm in length were implanted between the common iliac and external iliac artery in 12 adult dogs. The grafts were explanted between the first and the sixth months which followed the implantations. The healing process was observed by gross examination, microscopic and scanning electron microscopic examination. Prostaglandin PGE2, TXB2, 6 keto PGF1 alpha and PGF2 alpha were measured by radioimmunologic assay from samples retrieved from the medium part of the graft. During implantation, there was no notable difference in blood loss through the graft. At the time of explantation, 20 grafts were patent (10 CHG, 10 CG). In both grafts, the healing process developed progressively between 2 and 6 months and 90% of the internal surface of the grafts were covered with endothelial like cells. At 6 months, the internal layer was thinner in heparinized graft. PGI2 secretion was found with the two types of grafts. In conclusion, the present study showed no difference in the blood loss or healing characteristic of CHG and CG except for a potentially thinner internal layer with CHG. Comparative studies in humans are necessary to evaluate the potential benefit of heparin bonded graft in clinical practice.