Активность микрофлоры как показатель токсичности морских донных отложений шельфовой зоны Черного моря и Керченского пролива

Изучена потенциальная активность донной микрофлоры в местах утечки остатков химических токсикантов, затопленных в период Второй Мировой войны ХХ в. Отмечены особенности восстановления жизнедеятельности микрофлоры при различных уровнях загрязнения донных отложений мышьяком и хлорированными органическими сульфидами. Полученные результаты перспективно использовать при оценке экологического состояния донных отложений в загрязненных прибрежных акваториях

Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

<div><p>Background</p><p>A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension.</p><p>Principal Findings</p><p>Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. <i>In vitro</i>, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1).</p><p>Conclusion</p><p>These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.</p></div

Thrombin-induced nitric oxide-cGMP pathway dysfunction is mediated via activation of protease-activated receptor-1.

<p>Exposure of HUVECs to different concentration of the PAR-1 activating peptide TRAP-1 (1 to 100 µg/ml) over 6 hours leads to reduced expression of (A) eNOS, (B) sGC alpha 1 and (C) sGC beta 1, whereas (D) PDE5 protein expression is increased. (E) Prolonged TRAP-10 exposition over 14 hours leads to cGMP depletion in HUVECs in presence of DETA NONOate (100 µM). Data shown as mean ± SEM (A, B, C, D: n = 6/group; E: n = 9/group). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs cells not exposed to TRAP-10.</p

Thrombin modulates protein expression of key regulators of the nitric oxide-cGMP pathway in endothelial cells.

<p>Exposure of HUVECs to thrombin (30 nM) over 14 hours leads to a reduced expression of (A) eNOS, (B) sGC alpha 1 and (C) sGC beta 1, whereas (D) PDE5 protein expression is increased. Data shown as mean ± SEM (A, B, C: n = 5/group; D: n = 3/group). **<i>p</i><0.01, ***<i>p</i><0.001 vs cells not exposed to thrombin.</p

Thrombin modulates the expression of key regulators of the NO-cGMP pathway in a biphasic manner.

<p>Left panel: Short time stimulation of HUVECs with thrombin (50 nM) increases (A) eNOS and (E) sGC beta 1 expression. Right panel: By contrast, chronic stimulation decreases (B) eNOS and (D) sGC alpha 1 expression, whereas (H) PDE5 expression is increased. At late time points, all effects are concentration-dependent (B, D, H). (I) PDE5 is the only cGMP-hydrolyzing PDE to be upregulated by thrombin (30 nM). Data shown as mean ± SEM (A, C, E, G: n = 3–5/group; B, D, F, H: n = 11/group; I: n = 3/group). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs cells not exposed to thrombin.</p

cGMP-reducing effects of thrombin in the presence of pharmacological modulators of the nitric oxide-cGMP pathway.

<p>(A) Direct sGC-stimulator BAY 41–2272 (1 µM) und NO donor DETA NONOate (100 µM) elevate cGMP, but the prolonged thrombin challenge (0.3 nM) over 14 hours still reduces cGMP (white bars: controls; grey bars: thrombin). (B) When co-stimulated with DETA NONOate, both (B) Tadalafil and (C) IBMX elevate cGMP; again, however, cGMP-decreasing effects of thrombin were still present under stimulation with these agents. (D) The combination of IBMX and BAY 41–2272 increases cGMP more strongly than either compound alone, but thrombin-induced cGMP reduction is still present. Data shown as mean ± SEM (A: n = 5/group; B: n = 12/group; C: n = 3–5/group; D: n = 5/group). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Chronic thrombin stimulation leads to nitric oxide-cGMP pathway dysfunction in pulmonary endothelial cells.

<p>Thrombin (30 nM) stimulation over 6 hours reduces the expression of (A, E) eNOS, (B, F) sGC alpha 1 and (C, G) sGC beta 1 in pulmonary artery (A–D) and pulmonary microvascular (E–H) endothelial cells, whereas (D, H) PDE5 expression is increased (White bars: control cells; grey bars: with thrombin). (I) Prolonged thrombin exposition over 14 hours leads to cGMP depletion in pulmonary endothelial cells in presence of DETA NONOate (100 µM). Data shown as mean ± SEM (A, B, C, D: n = 6/group; E, F, G, H: n = 12/group; I: n = 5–6). *<i>p</i><0.05, **<i>p</i><0.01 vs cells not exposed to thrombin.</p

Thrombin has biphasic effects on cGMP levels in endothelial cells.

<p>(A) Thrombin (30 nM) acutely increases cGMP content in HUVECs; the effects, however, are transient (white bars: controls; grey bars: with thrombin). (B) cGMP elevation by incubation with the NO donor DETA NONOate (100 µM) is partly blunted by thrombin over 14 to 24 hours. (C) Reduction of cGMP levels in DETA NONOate-stimulated cells is concentration-dependent. (D) Thrombin-induced reduction of cGMP levels over 14 hours in DETA NONOate-stimulated cells is completely abolished by direct thrombin inhibitor melagatran (10 µM). Data shown as mean ± SEM (A, B, C: n = 3; D: n = 4/group). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs cells not exposed to thrombin.</p

Marginal effects of FXa on key regulators of the nitric oxide-cGMP pathway in endothelial cells.

<p>Incubation of HUVECs with FXa for 6 hours reduces eNOS (A) and increases PDE5 (D) mRNA expression significantly only at the highest concentration of 30 nM. No effect on sGC alpha 1 (B) and sGC beta 1 (C) is detectable. Data shown as mean ± SEM (n = 6/group). *<i>p</i><0.05, ***<i>p</i><0.001 vs cells not exposed to thrombin.</p

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII