Zoltan Veresh

ADMA Impairs Nitric Oxide–Mediated Arteriolar Function Due to Increased Superoxide Production by Angiotensin II–NAD(P)H Oxidase Pathway

Asymmetrical dimethylarginine (ADMA) is thought to be an endogenous regulator of arteriolar tone by inhibiting NO synthase. However, our previous studies showed that, in isolated arterioles, ADMA induced superoxide production as well. Thus, the mechanisms by which ADMA affects arteriolar tone remain obscure. We hypothesized that ADMA, by activating NAD(P)H oxidase, increases superoxide production, interfering with NO mediation of flow-induced dilation. In the presence of indomethacin, isolated arterioles from rat gracilis muscle (≈160 &mgr;m at 80 mm Hg) were incubated with ADMA (10−4 mol/L), which elicited significant constriction (from 162±4 to 143±4 &mgr;m) and eliminated the dilations to increases in intraluminal flow (from a maximum 31±2% to 3±1%; P<0.05). In the presence of ADMA, superoxide dismutase plus catalase restored dilations to flow (from a maximum 3±1% to 28±2%). Endothelial denudation or incubation of arterioles with the NAD(P)H oxidase inhibitor apocynin or the angiotensin-converting enzyme inhibitor quinapril inhibited ADMA-induced constriction. In addition, apocynin, quinapril, or the angiotensin type 1 receptor blocker losartan restored flow-induced dilations reduced by ADMA. Furthermore, inhibition of NO synthase abolished the “superoxide dismutase/catalase-restored” flow-induced dilation in the presence of ADMA. ADMA-induced increased production of superoxide, assessed by dihydroethidium fluorescence, was inhibited by apocynin, quinapril, or losartan. We suggest that ADMA activates the local renin-angiotensin system, and the angiotensin II released activates NAD(P)H oxidase; superoxide produced interferes with the bioavailability of NO, resulting in diminished flow-induced dilation, a mechanism that may contribute to the development of arteriolar dysfunction and increased tone associated with elevated ADMA levels.

The Frequency of Peripheral Blood CD4+ CD25high FoxP3+ and CD4+ CD25− FoxP3+ Regulatory T Cells in Normal Pregnancy and Pre‐Eclampsia

Regulatory T cells (Tregs) play an important role in the development of pregnancy‐specific immune tolerance. We aimed to determine the peripheral frequency of a recently described Treg subpopulation, the CD4+ CD25− FoxP3+ Treg subset, and its correlation with the conventional CD4+ CD25high FoxP3+ Tregs in normal pregnancy (NP) and pre‐eclampsia (PE) compared to non‐pregnant (non‐P) women. We also examined the proportion of the activated CD4+ CD25high FoxP3high Treg subset within conventional Treg cells.

Evaluation of a rapid and simple placental growth factor test in hypertensive disorders of pregnancy.

The aim of this study was to investigate the diagnostic accuracy of the Triage placental growth factor (PlGF) assay, together with its prognostic efficiency in determining the need for preterm delivery in all forms of hypertensive disorders of pregnancy. A total of 130 pregnant women with a diagnosis of preeclampsia (PE: 23), HELLP syndrome (20), superimposed preeclampsia (SIPE: 17), chronic hypertension (CHT: 25), gestational hypertension (GHT: 18) and 27 normotensive pregnant controls were enrolled in this case-control study. A single blood sample was taken between 22 and 34 weeks of gestation, and the plasma was analyzed for PlGF using the Alere Triage PlGF assay. The PlGF levels found in all hypertensive disorder groups differed significantly from those observed in controls. There was a highly significant difference in PlGF concentrations between women with a pregnancy duration <35 weeks and controls. Using a gestational age-dependent threshold of 5% of normal, a positive PlGF test predicted delivery before 35 weeks in 93.7% of hypertensive women and delivery before 37 weeks in 90.5% of hypertensive women. A positive PlGF test identified the following proportions of hypertensive patients: 95.7% (PE), 95.0% (HELLP syndrome), 82.4% (SIPE), 60.0% (CHT) and 44.4% (GHT). A positive PlGF test was associated with a significantly shorter duration of pregnancy (hazard ratio of 3.43 adjusted for the gestational age at the time of sample collection and hypertension with proteinuria). In conclusion, PlGF concentrations are significantly lower in all hypertensive disorders. A positive test using the Triage PlGF assay at 22–34 weeks of gestation predicts delivery before 37 weeks in women with both proteinuric and non-proteinuric hypertensive disorders of pregnancy.

Circulating levels of the anti‐angiogenic thrombospondin 2 are elevated in pre‐eclampsia

An imbalance of maternal circulating pro‐ and anti‐angiogenic factors may play a role in the pathogenesis of pre‐eclampsia. Thrombospondin 2 (TSP‐2) is a protein expressed mainly by activated endothelial cells, which acts as a potent anti‐angiogenic agent. Our aim was to determine whether serum TSP‐2 levels are altered in pre‐eclampsia. We enrolled 35 pre‐eclamptic patients and 35 healthy pregnant women in the study. Thrombospondin 2 levels were determined by enzyme‐linked immunosorbent assay, while soluble fms‐like tyrosine kinase‐1 (sFlt‐1) and placental growth factor (PlGF) concentrations were determined by electrochemiluminescence immunoassay. In patients with PE, we demonstrated 1.7‐fold higher TSP‐2 [13.2 (9.4–18.1) vs. 7.9 (7.2–11.2) ng/ml, p<0.001], 3.8‐fold higher sFlt‐1 and 4.3‐fold lower PlGF levels compared with the control group. There were no associations between TSP‐2 and sFlt‐1 or PlGF concentrations. We suggest that circulating TSP‐2 levels may contribute to the pathogenesis of PE via its anti‐angiogenic properties, but in a distinct way from sFlt‐1 and PlGF.

Asymmetric Dimethylarginine Reduces Nitric Oxide Donor-Mediated Dilation of Arterioles by Activating the Vascular Renin-Angiotensin System and Reactive Oxygen Species

Introduction: We tested the hypothesis that asymmetric dimethylarginine (ADMA) interferes with other mechanisms in addition to inhibition of nitric oxide synthase (NOS). Thus, in skeletal muscle arterioles, in the presence of ADMA, we investigated the dilator effect of an NO donor and increases in flow and aimed to elucidate the underlying mechanisms, including the role of oxidative stress, which is known to reduce the bioavailability of NO. Methods and Results: In isolated rat gracilis skeletal muscle arterioles (∼160 µm at 80 mm Hg), ADMA (similarly to pyrogallol) reduced dilations to sodium nitroprusside (SNP), which was significantly prevented by the presence of superoxide dismutase (SOD) and catalase (CAT): SNP 10–8M; control: 43.2 ± 3%, ADMA: 4.9 ± 1%, ADMA + SOD/CAT: 30.2 ± 9% (p < 0.05). Also, ADMA reduced basal diameter and flow-induced dilations, which were not restored by L-arginine, but prevented by SOD/CAT and by inhibition of NAD(P)H oxidase (but not xanthine oxidase) and by an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker (ARB). ADMA increased the production of reactive oxygen species detected by lucigenin-enhanced chemiluminescence, which was significantly inhibited by SNP or ARB. Conclusion: We suggest that by activating the vascular renin-angiotensin-NAD(P)H oxidase pathway, ADMA elicits oxidative stress, which interferes with the bioavailability of NO and consequently reduces NO-mediated dilations.

Thromboxane A2 Contributes to the Mediation of Flow-Induced Responses of Skeletal Muscle Venules: Role of Cyclooxygenases 1 and 2

Background: It has been shown that increases in intraluminal flow elicit dilation in venules, but the mediation of response is not yet clarified. We hypothesized that – in addition to nitric oxide (NO) and dilator prostaglandins (PGI2/ PGE2) – thromboxane A2 (TxA2) contributes to the mediation of flow-induced responses of venules. Methods and Results: Isolated rat gracilis muscle venules (259 ± 11 μm at 10 mm Hg) dilated as a function of intraluminal flow, which was augmented in the presence of the TxA2 receptor antagonist SQ 29,548 or the TxA2 synthase inhibitor ozagrel. In the presence of SQ 29,548, indomethacin or Nω-nitro-L-arginine methyl-ester decreased flow-induced dilations, whereas in their simultaneous presence dilations were abolished. The selective cyclooxygenase (COX) 1 inhibitor SC 560 reduced, whereas the selective COX-2 inhibitor NS 398 enhanced flow-induced dilations. Immunohistochemistry showed that both COX-1 and COX-2 are present in the wall of venules. Conclusion: In skeletal muscle venules, increases in intraluminal flow elicit production of constrictor TxA2, in addition to the dilator NO and PGI2/PGE2, with an overall effect of limited dilation. These mediators are likely to have important roles in the multiple feedback regulation of wall shear stress in venules during changes in blood flow velocity and/or viscosity.

Hydrogen peroxide via thromboxane A2 receptors mediates myogenic response of small skeletal muscle veins in rats.

UNLABELLED We aimed to test two hypotheses: (1) isolated small veins develop substantial myogenic tone in response to elevation of intraluminal pressure, (2) H2O2 contributes to the mediation of myogenic response via activation of arachidonic acid (AA) cascade and release constrictor prostaglandins. METHODS Small veins were isolated from gracilis muscle of male rats, then cannulated. Changes of diameter to increases in intraluminal pressure, H2O2 and arachidonic acid in the presence and absence of various inhibitors were measured by videomicroscope and microangiometer. At the end of experiments the passive diameter were obtained in Ca2+ -free PSS solution. RESULTS Isolated rat gracilis muscle small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (1-12 mmHg). Calculated maximum myogenic tone was 70 ± 5% at 10 mmHg. Presence of catalase or indomethacin or SQ 29,548 reduced significantly the pressure-induced myogenic response. Also, H2O2 (10-9-10-5 M) and arachidonic acid (10-7-10-4 M) elicited concentration dependent constrictions, which were inhibited by the presence of indomethacin or SQ 29,548. CONCLUSION We propose that both myogenic response and pressure-induced release of H2O2 play important roles in regulating the vasomotor function of venules both in physiological and pathological conditions.

OS085. Decreased maternal circulating PLGF is a significant predictor of length of pregnancy in women with hypertensive disorders of pregnancy.

INTRODUCTION Diagnosis of the presence of disease and prediction of the rate of progression of disease in women with hypertensive disorders of pregnancy remains a clinical problem. Better methods are needed to determine the magnitude of risk to support patient counseling and clinical management. OBJECTIVES To investigate whether the level of free PlGF is a significant predictor of length of pregnancy in women with hypertension. METHODS In this case-control study a single sample was taken between the 22nd and 34th completed gestational weeks from 130 pregnant women with a final diagnosis of: pre-eclampsia (PE), HELLP-syndrome, superimposed pre-eclampsia (SIPE), chronic hypertension (CHT), gestational hypertension (GHT), and normal healthy pregnancy (Control). Plasma was analysed for PlGF using the Triage® PlGF assay (Alere, San Diego). A positive PlGF test was defined as below the 5th centile of normal healthy pregnancy. Hazard ratios for length-of-pregnancy were calculated for a positive PlGF test in a multivariate Cox proportional hazards model adjusting for two covariates, the gestational age at sample collection and a final diagnosis of proteinuric hypertension (PE, HELLP, and SIPE). RESULTS Median PlGF concentration was significantly lower in women with hypertension than in controls. Women with proteinuric hypertension had the lowest levels of PlGF. A positive PlGF test predicted delivery before 35 weeks in 93.7% women, and delivery before 37 weeks in 90.5% women. A positive PlGF test was associated with a significantly higher risk of imminent delivery. PlGF was a significant and independent predictor of women destined to deliver early because of maternal or fetal complication (adjusted Hazard Ratio of 3.43, 95%CI of 1.97 to 5.98). CONCLUSION A positive PlGF test is significant predictor of length of pregnancy, independent of other diagnostic criteria. PlGF has the potential to identify increased risk without the limitation of non-specificity which exists with other diagnostic parameters.

PP056. Placental growth factor is a better predictor of preterm birth than uterine or umbilical artery doppler in hypertensive disorders of pregnancy.

INTRODUCTION Diagnosis of the presence of disease and prediction of the rate of progression of disease in women with hypertensive disorders of pregnancy remains a clinical problem. OBJECTIVES Adequate placentation and placental development are important for normal pregnancy. We determined whether PlGF is a better predictor of delivery before 34+0 and 37+0 weeks than uterine artery and umbilical artery doppler. METHODS One hundred and four women presenting before the completed 34th week of pregnancy with hypertensive disorders of pregnancy were enrolled into the study. Final diagnosis was chronic hypertension (N=24), gestational hypertension (N=21), pre-eclampsia (N=24), HELLP-syndrome (N=15), superimposed preeclampsia (N=20). Blood draw occurred before the 34th week of pregnancy at the time of investigation for expedited delivery or expectant management. Plasma was analysed for PlGF by the Alere Triage® PlGF assay using fluorescently-labelled monoclonal antibodies against PlGF. A positive PlGF test was defined as below the 5th centile of normal healthy pregnancy. RESULTS Of the 104 pregnant women, the level of PlGF was abnormal in 23 of 24 (96%) women with IUGR, compared to 10 of 24 (42%) and 14 of 24 (58%) for uterine artery doppler and umbilical artery doppler, respectively. In the case of pre-eclampsia, the level of PlGF was abnormal in 50 of 59 (85%), compared to 15 of 59 (25%) and 25 of 56 (45%) for uterine artery doppler and umbilical artery doppler, respectively. Forty four (42%) women required medical delivery before 34+0 weeks gestation and 68 (65%) before 37+0 weeks gestation (see Table). PlGF detected a higher number of women requiring early delivery than doppler. CONCLUSION The new Triage® PlGF test provides useful information to inform clinical decisions in pregnancy-associated hypertensive disorders, before the 34th completed gestational week.