Experimental Pulmonary Embolism Research Articles

The Reciprocity of M- and N-Cholinergic Mechanisms in the Pulmonary Microcirculatory Changes in Case of Experimental Pulmonary Embolism

The Reciprocity of M- and N-Cholinergic Mechanisms in the Pulmonary Microcirculatory Changes in Case of Experimental Pulmonary Embolism

Terlipressin Increases Systemic and Lowers Pulmonary Arterial Pressure in Experimental Acute Pulmonary Embolism.

We investigated whether the vasopressin-analog, terlipressin induces systemic vasoconstriction and pulmonary vasodilation in a porcine model of acute pulmonary embolism. Controlled, animal study. Tertiary medical center research laboratory. Female pigs (n = 12, Cross of Land Race, Duroc, and Yorkshire ~ 60 kg). Acute pulmonary embolism was induced by administration of three large autologous emboli. Animals then received four increasing doses of either terlipressin (n = 6) or vehicle (n = 6). Effects were evaluated in vivo at baseline, after pulmonary embolism and after each dose by invasive hemodynamic measures, transesophageal echocardiography, and blood analysis. Isolated pulmonary arteries were evaluated ex vivo in a myograph. Pulmonary embolism caused a four-fold increase in pulmonary vascular resistance (p < 0.0001) and a two-fold increase in mean pulmonary arterial pressure (p < 0.0001) compared with baseline. Terlipressin increased mean systemic blood pressure (28 ± 5 mm Hg; p < 0.0001) and systemic vascular resistance (1,320 ± 143 dynes; p < 0.0001) compared with vehicle. In the pulmonary circulation, terlipressin decreased mean pulmonary arterial pressure (-6.5 ± 1.8 mm Hg; p = 0.005) and tended to decrease pulmonary vascular resistance (-83 ± 33 dynes; p = 0.07). Terlipressin decreased cardiac output (-2.5 ± 0.5 L/min; p < 0.0001) and increased plasma lactate (2.7 ± 0.2 mmol/L; p < 0.0001), possibly indicating systemic hypoperfusion. A biomarker of cerebral ischemia, S100b, remained unchanged, suggesting preserved cerebral perfusion (0.17 ± 0.11 µg/L; p = 0.51). Ex vivo, terlipressin relaxed pulmonary and constricted mesenteric arteries. Terlipressin caused systemic vasoconstriction and pulmonary vasodilation in a porcine in vivo model of acute pulmonary embolism and vasorelaxation in isolated pulmonary arteries. Despite positive vascular effects, cardiac output declined and plasma lactate increased probably due to a predominantly systemic vasoconstrictor effect of terlipressin. These findings should warrant careful translation to the clinical setting and does not suggest routine use in acute pulmonary embolism.

Treatment with new organic nitrites in pulmonary hypertension of acute experimental pulmonary embolism.

Acute pulmonary embolism may cause right heart failure due to increased pulmonary vascular resistance and arterial hypoxemia. Effective vasodilator therapy of the pulmonary hypertension is highly needed. Therefore, we investigated the effects of a newly developed effective pulmonary vasodilator, the organic mononitrites of 1,2‐propanediol (PDNO), in a rabbit model of acute pulmonary embolism. In anesthetized and ventilated rabbits, systemic and pulmonary hemodynamics, exhaled nitric oxide (NO), plasma nitrite concentration, and blood gases were monitored. First, dose–response experiments with intravenous and left heart ventricle infusions of PDNO and inorganic nitrite were done in naive animals and in pulmonary hypertension induced by a thromboxane A2 analogue. Second, acute pulmonary embolism was induced and either PDNO or placebo were administered intravenously within 20 minutes and evaluated within 1 hour after pulmonary embolization. PDNO intravenously, in contrast to inorganic nitrite intravenously, increased exhaled NO and counteracted pulmonary hypertension and vasodilated the systemic circulation, dose‐dependently, thereby showing efficient NO donation. Pulmonary embolization induced pulmonary hypertension and gas exchange disturbances. PDNO significantly decreased and normalized pulmonary vascular resistance and the right ventricle rate‐pressure product, without causing tolerance, with no significant side effects on the systemic circulation, nor on blood‐gas values or on methemoglobin formation. In conclusion, PDNO is a NO donor and an efficient vasodilator in the pulmonary circulation. Treatment with this or similar organic nitrites intravenously may be a future option to avoid right heart failure in life‐threatening acute pulmonary embolism.

АЛЬФА-АДРЕНЕРГИЧЕСКИЕ МЕХАНИЗМЫ КОНСТРИКТОРНЫХ РЕАКЦИЙ ЛЕГОЧНЫХ ВЕН ПРИ ЭКСПЕРИМЕНТАЛЬНОЙ ТРОМБОЭМБОЛИИ ЛЕГОЧНОЙ АРТЕРИИ, "Российский физиологический журнал им. И.М. Сеченова"

В острых опытах на 30 кроликах изучали изменения легочной гемодинамики при моделировании тромбоэмболии легочной артерии в условиях двухканальной перфузии изолированных легких постоянным объемом, а также целостного кровообращения в контроле и на фоне блокады a-адренорецепторов фентоламином. Тромбоэмболия легочной артерии при перфузии легких приводила к повышению давления в легочной артерии, легочного сосудистого сопротивления и прекапиллярного сопротивления. У животных также отмечено возрастание среднего капиллярного гидростатического давления, коэффициента капиллярной фильтрации и посткапиллярного (венозного) сопротивления. При тромбоэмболии легочной артерии на фоне блокады a-адренорецепторов давление в легочной артерии, капиллярное гидростатическое давление, легочное сосудистое сопротивление, прекапиллярное и посткапиллярное сопротивления возрастали меньше, чем в контроле, а коэффициент капиллярной фильтрации повышался в большей степени. В случае тромбоэмболии легочной артерии в условиях целостного кровообращения в контроле сердечный выброс уменьшался более выраженно, чем легочный кровоток; кровоток по краниальной полой вене снижался меньше, чем по каудальной. При тромбоэмболии на фоне блокады a-адренорецепторов указанные показатели снижались примерно на одинаковую величину. Полученные данные свидетельствуют об участии a-адренергических механизмов в констриктор- ных реакциях не только артериальных, но и венозных сосудов легких в условиях тромбоэмболии легочной артерии.

Expression and Correlation of Hypoxia-Inducible Factor-1α (HIF-1α) with Pulmonary Artery Remodeling and Right Ventricular Hypertrophy in Experimental Pulmonary Embolism.

BackgroundWe investigated the expression of HIF-1α (hypoxia-inducible factor-1α) and its correlations with pulmonary artery remodeling and right ventricular hypertrophy in PE (pulmonary embolism) rats.Material/MethodsAfter the PE rat model was established, the dynamic changes of mPAP (mean pulmonary artery pressure), PAMT (relative medial thickness of small pulmonary arteries), WA/TA (vessel wall area/total area), and RVHI (right ventricular hypertrophy index) were detected. Then, histomorphology of pulmonary vascular was observed, followed by HIF-1α mRNA and protein for pulmonary artery and right ventricle were checked via in situ hybridization and immunohistochemistry, respectively. The correlations between HIF-1α and mPAP, PAMT, WA/TA, and RVHI were analyzed.ResultsThe mPAP level increased from the initial time to 12 weeks of PE and reached the peak at 12 weeks. After 4 weeks of PE, PAMT increased compared with the initial control group (p<0.05), and increased further after 8 weeks and 12 weeks. Changes of the vessel WA/TA were the same as PAMT. Compared with the initial control group, RVHI increased at 8 weeks (p<0.05) and 12 weeks of PE (p<0.01). HIF-1α mRNA and protein were positively correlated with mPAP, PAMT, and WA/TA in pulmonary arteries. HIF-1α mRNA and protein were positively correlated with mPAP, PAMT, WA/TA, and RVHI in right ventricles (p<0.01).ConclusionsHIF-1α mRNA and protein are expressed in the pulmonary artery and right ventricular of PE rats, and HIF-1α may correlate with pulmonary artery remodeling and right ventricular hypertrophy in rats with PE.

Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli

Background: In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding. Methods: Pulmonary embolism was induced by jugular vein infusion of 125 I-fibrin or fluorescein isothiocyanate-fibrin labeled emboli in anesthetized mice. Thrombus site expression of key fibrinolytic molecules was determined by immunofluorescence staining. The effects of r-tPA and α2-antiplasmin inactivation on fibrinolysis and bleeding were examined in a humanized model of pulmonary embolism. Results: The plasminogen activation and plasmin inhibition system assembled at the site of acute pulmonary emboli in vivo. Thrombus dissolution was markedly accelerated in mice with normal α2-antiplasmin levels treated with an α2-antiplasmin–inactivating antibody ( P &lt;0.0001). Dissolution of pulmonary emboli by α2-antiplasmin inactivation alone was comparable to 3 mg/kg r-tPA. Low-dose r-tPA alone did not dissolve emboli, but was synergistic with α2-antiplasmin inactivation, causing more embolus dissolution than clinical-dose r-tPA alone ( P &lt;0.001) or α2-antiplasmin inactivation alone ( P &lt;0.001). Despite greater thrombus dissolution, α2-antiplasmin inactivation alone, or in combination with low-dose r-tPA, did not lead to fibrinogen degradation, did not cause bleeding (versus controls), and caused less bleeding than clinical-dose r-tPA ( P &lt;0.001). Conclusions: Although the fibrinolytic system assembles at the site of pulmonary emboli, thrombus dissolution is halted by α2-antiplasmin. Inactivation of α2-antiplasmin was comparable to pharmacological r-tPA for dissolving thrombi. However, α2-antiplasmin inactivation showed a unique pattern of thrombus specificity, because unlike r-tPA, it did not degrade fibrinogen or enhance experimental bleeding. This suggests that modifying the activity of a key regulator of the fibrinolytic system, like α2-antiplasmin, may have unique therapeutic value in pulmonary embolism.

実験的肺脂肪塞栓症の病理 : 中性脂肪または脂肪酸静注を受けたウサギ肺の形態学的変化, 特に肺脂肪塞栓症の発生病理に関する考察

実験的肺脂肪塞栓症の病理 : 中性脂肪または脂肪酸静注を受けたウサギ肺の形態学的変化, 特に肺脂肪塞栓症の発生病理に関する考察

Peculiarities of Blood Flow Changes in Venae Cavae during Experimental Pulmonary Embolism.

The model of acute pulmonary embolism in rabbits demonstrated reduced pulmonary blood flow, cardiac output, left atrial pressure, and blood flow in venae cavae against the background of elevated left pulmonary artery pressure and increased pulmonary vascular resistance. Simultaneously, the blood flow in the superior vena cava decreased to a lesser extent than that in the inferior vena cava, which was a characteristic feature of the model of pulmonary pathology. In contrast, when histamine was infused into the left jugular vein to equally elevate pressure in pulmonary artery as in the above model, the blood flow in the superior vena cava decreased to a greater extent than that in inferior vena cava. During stenosis of inferior vena cava that decreased the cardiac output to the level observed during modeled pulmonary embolism, the blood flows in both venae cavae dropped equally.

Abstract 16486: Safely Removing the Brakes on Fibrinolysis: Inactivation of α2-Antiplasmin Dissolves Pulmonary Emboli Without Causing Bleeding

Introduction: Venous thromboembolism affects millions of patients per year and pulmonary embolism (PE) is a leading cause of hospital mortality. PE activates the fibrinolytic system, but regulatory molecules such as α2-antiplasmin (a2AP) appear to prevent thrombus dissolution. Pharmacologic tissue plasminogen activator (tPA) dissolves pulmonary emboli, relieves right heart strain and improves mortality in high risk patients, but tPA has unacceptable bleeding risks. We examined whether inactivation of a2AP may prove to be a safer and more effective method for dissolving experimental pulmonary emboli. Methods: Pulmonary embolism was examined in a humanized model. Anesthetized, a2AP -/- mice, supplemented with physiological levels of a2AP, were infused with 125 I-labeled human thrombi via the jugular vein. Then mice were treated with tPA, no agent (control), a humanized chimeric monoclonal antibody inactivator of a2AP (a2AP-I) or the combination of low dose tPA and a2AP-I. Thrombus dissolution was determined by gamma counting and surgical bleeding was measured. Results: Clinical dose tPA dissolved pulmonary emboli more effectively than no therapy (control, p&lt;0.001). However, mice treated with a2AP-I showed greater thrombus dissolution than tPA (p&lt;0.001) or controls (p&lt;0.001). Alone, low doses of tPA did not dissolve thrombi, but in combination with a2AP-I, low dose tPA increased thrombus dissolution more than clinical dose tPA alone (p&lt;0.001) or a2AP-I alone (p&lt;0.001). Clinical dose tPA therapy was associated with a significant increase in bleeding (p&lt;0.01). Though more potent than tPA in dissolving pulmonary emboli, a2AP-I alone, or in combination with low dose tPA, did not increase bleeding risk vs. controls and caused less bleeding than tPA (p&lt;0.01). Conclusion: In experimental pulmonary embolism a2AP plays a critical regulatory role in blocking fibrinolysis. Inactivation of a2AP was more potent than tPA in dissolving thrombi in PE, but unlike tPA, it did not cause bleeding. This suggests that a2AP inactivation may be a safer strategy for treating venous thromboembolism.

Effect of combined treatment with urokinase and endothelin-1 receptor antagonist on myocardial perfusion in experimental acute pulmonary embolism

Objective To observe the effect of the selective endothelin receptor antagonist BQ-123 combined with urokinase against myocardial injury due to acute pulmonary embolism (APE).Methods Twenty healthy adult Beagle dogs were used,and randomly assigned to five groups:sham-operated group (sham group),pulmonary embolism group (PE group),urokinase thrombolysis grouop (UK group),BQ123 group (BQ group) and urokinase + BQ-123 combination group (UK + BQ group),with 4 dogs in each group.The parameters of the mean pulmonary artery pressure (mPAP),and coronary blood flow (CBF) at different time pointi pre-and post-embolization were recorded and compared,and the serum cTnT level at 6 h post-embolization was determined.Results (1) After pulmonary embolism,mPAP was obviously increased,and at 4 h,mPAP in UK group and UK + BQ group was decreased to (26.67 ± 2.08) and (20.00 ± 2.00) mmHg (1 mmHg =0133 kPa) respectively,remarkably lower than that in PE group (P ＜ 0.05) ; (2) The serum cTnT level at 6 h post-embolization in UK ± BQ group [(611.93 ±97.50) ng/L] was significantly lower than that in UK group [(747.22 ± 62.72) ng/L] and PE group [(900.79 ± 71.11) ng/L] (P ＜0.05) ; (3) The CBF in PE group was obviously declined,and in UK group,BQ group and UK + BQ group,the CBF was increased to varying degrees.UK + BQ group had the greatest increase of CBF and showed statistically significant difference from the other two groups at different time points post-embolization (P ＜ 0.05).Conclusion On the basis of thrombolysis therapy,endothelin-1 receptor antagonist BQ-123 can increase the CBF significantly and relieve the acute myocardial ischemia induced by APE. Key words: Pulmonary embolism ; Endothelin receptor antagonist ; Thrombolysis ; Troponin T; Myocardial ischemia

The effect of low molecular heparin and urokinase on MCP-1 of acute experimental pulmonary embolism in rabbits

To evaluate effect the of thrombolytic (urokinase, UK) and anticoagulant agent (low-molecular-weight heparin, LMWH) on the pulmonary injury of rabbits with acute pulmonary embolism (PE) by assaying monocyte chemoattractant protein-1 (MCP-1). Rabbit models with PE were established by transfusing autologous blood clots on 60 healthy male Japanese white rabbits. Experimental PE rabbits were randomly divided into 3 groups:normal saline (NS) group (n = 18) , LMWH group (n = 18) and UK group (n = 18), and other 18 rabbits underwent sham operations as SHAM group (n = 18). Each group was divided into 3 subgroups based on 2 days (day 2), 4 days (day 4), and 14 days (day 14) after therapies. Arterial blood gas analysis was measured. MCP-1 levels in lung tissue and blood were assayed with ELISA at various times (day 2, day 4 and day 14 ). Fixed sections were stained with trichrome for intimal hyperplasia determination. The overall rate of success for making PE rabbit models was 90% (54/60), which was not affected by treatment. Compared with NS group, P(A-a)O2 significantly decreased in UK group. Compared with NS group, MCP-1 levels in lung tissue significantly decreased in LMWH group on day 4 [(33 ± 9) ng/L vs (48 ± 5) ng/L, P < 0.05] and day 14 [(30 ± 11) ng/L vs (41 ± 4) ng/L, P < 0.05]; MCP-1 levels in serum on day 14 also significantly decreased in LMWH group [(36 ± 10) ng/L vs (51 ± 5) ng/L, P < 0.05]. Compared with NS group, MCP-1 levels in lung tissue significantly decreased in UK group on day 2 and 4 [Day 2: (34 ± 8) ng/L vs (50 ± 4) ng/L, P < 0.05; Day 4: (29 ± 7) ng/L vs (48 ± 5) ng/L, P < 0.05]; MCP-1 levels in serum on day 2 and day 4 also significantly decreased in UK group [Day 2: (44 ± 3) ng/L vs (48 ± 3) ng/L, P < 0.05; Day 4: (44 ± 4) ng/L vs (53 ± 1)ng/L, P < 0.05]. UK treatment may rapidly improve V/Q ratio and decrease MCP-1 levels in lung tissue or serum, but it can not inhibit persistent inflammation. LMWH can decrease MCP-1 levels in lung tissue or serum, and inhibit persistent inflammation and late intimal hyperplasia.

Interview with Donald E Ingber.

Interview with Donald E Ingber.

Expression of tissue factor in pulmonary artery after experimental acute pulmonary embolism

Objective To investigate the expression of tissue factor and explore its clinical significances in pulmonary artery after acute pulmonary thromboembolism.Methods Thirty-four Japanese white rabbits (Level Ⅱ animals) were randomly (random number) assigned into four groups:group A (specimen of pulmonary artery was taken 3 hours after pulmonary embolism,n =8),group B (specimen of pulmonary artery was taken 8 hours after pulmonary embolism,n =8),group C (specimen of pulmonary artery was taken 24 hours after pulmonary embolism,n =8) and control group (pseudo-operations were carried out without injecting autologous blood clots,n =10).The animal model of pulmonary thromboembolism was established by injecting autologous blood clots into jugular vein through a 5F catheter and confirmed by digital subtraction angiography.The mRNA expression of TF in different parts of pulmonary artery was assayed by RT-PCR.The q test was utilized if there was a significant difference in a given continuous variable among three groups analyzed by ANOVA.Results The TF expression in the specimen adjacentto emboli was stable at 3 h,8 h or 24 hours after embolism.The mRNA expression of TF at 3 h and 8 h after embolism was lower in specimen taken from distal-end of morbid pulmonary artery than those adjacent to emboli.While at 24 hours after embolism,there were similar mRNA expressions in specimen either adjacent or distal to emboli.Conclusions The high expression of tissue factor in pulmonary artery tissue adjacent to emboli could lead to locally increased coagulation activity,indicating the necessity of initiating anti-coagulation treatment as soon as possible after acute pulmonary embolism. Key words: Pulmonary Embolism; Tissue Factor; Pulmonary; Gene Expression; Thrombus; Anticoagulation ; Thrombolysis ; Rabbits

In vivo roles of factor XII

Coagulation factor XII (FXII, Hageman factor, EC = 3.4.21.38) is the zymogen of the serine protease, factor XIIa (FXIIa). FXII is converted to FXIIa through autoactivation induced by "contact" to charged surfaces. FXIIa is of crucial importance for fibrin formation in vitro, but deficiency in the protease is not associated with excessive bleeding. For decades, FXII was considered to have no function for coagulation in vivo. Our laboratory developed the first murine knockout model of FXII. Consistent with their human counterparts, FXII(-/-) mice have a normal hemostatic capacity. However, thrombus formation in FXII(-/-) mice is largely defective, and the animals are protected from experimental cerebral ischemia and pulmonary embolism. This murine model has created new interest in FXII because it raises the possibility for safe anticoagulation, which targets thrombosis without influence on hemostasis. We recently have identified platelet polyphosphate (an inorganic polymer) and mast cell heparin as in vivo FXII activators with implications on the initiation of thrombosis and edema during hypersensitivity reactions. Independent of its protease activity, FXII exerts mitogenic activity with implications for angiogenesis. The goal of this review is to summarize the in vivo functions of FXII, with special focus to its functions in thrombosis and vascular biology.

Pulmonary vascular reserve during experimental pulmonary embolism: Effects of a soluble guanylate cyclase stimulator, BAY 41-8543*

Pulmonary embolism causes pulmonary hypertension by mechanical obstruction and vasoconstriction. Therapeutic potential of pharmacologic dilation of unblocked vessels has received limited attention. We tested pulmonary vasodilator reserve using a soluble guanylate cyclase stimulator, BAY 41-8543. Controlled animal study. Medical center research laboratory. Male Sprague-Dawley rats. Pulmonary embolism was induced by infusing 25-μm plastic microspheres in the right jugular vein, producing mild or moderate pulmonary hypertension. Control animals with no pulmonary embolism received suspension medium for microspheres. Mild pulmonary embolism increased right ventricular peak systolic pressure (from 28 to 38 mm Hg) and decreased cardiac output (from 46 to 34 mL/min) with no change in mean arterial pressure. Infusion of BAY 41-8543 (50-200 μg/kg) decreased right ventricular peak systolic pressure. Five hrs moderate pulmonary embolism increased right ventricular peak systolic pressure (from 28 to 47 mm Hg) and decreased cardiac output (from 48 to 27 mL/min), causing right ventricular peak systolic pressure/cardiac output to increase from 0.6 control with no pulmonary embolism to 1.8 mm Hg/mL/min in 5-hr moderate pulmonary embolism + solvent for BAY 41-8543. Treatment of 5-hr moderate pulmonary embolism with BAY 41-8543 (50 μg/kg) caused a 2.2-fold increase in cardiac output (59 mL/min) with a 46% reduction in right ventricular peak systolic pressure (38 mm Hg), suggesting significant pulmonary vasodilation. Moderate pulmonary embolism decreased arterial sO2 (from 83% to 71%) and increased lactate (from 0.5 to 2.3 mmol/L). Treatment with BAY 41-8543 normalized sO2 and lactate. Hemolysis occurred during moderate experimental pulmonary embolism (60-fold increase in plasma hemoglobin). Treatment with BAY 41-8543 reduced free plasma hemoglobin content by 80%. In the setting of moderate impervious pulmonary embolism, treatment with a guanylate cyclase stimulator normalized pulmonary hemodynamics, reduced hemolysis, and improved oxygenation. These data support the hypothesis that pharmacologic dilation of nonobstructed pulmonary vasculature can effectively treat acute pulmonary hypertension from pulmonary embolism.

Arginase depletes plasma l-arginine and decreases pulmonary vascular reserve during experimental pulmonary embolism

The experiments test if experimental PE causes red blood cell hemolysis, arginase release and depletion of l-arginine and determine if arginase inhibition preserves l-arginine and improves pulmonary hemodynamics during PE. Experimental PE was induced in male Sprague–Dawley rats by infusing 25 μm microspheres (1.8 million/100 g body wt) in the jugular vein, producing moderate pulmonary hypertension. Pulmonary vascular resistance was estimated from the quotient of the right ventricular peak systolic pressure/cardiac output. Arterial plasma hemoglobin (ELISA), arginase activity (colorimetric assay) and l-arginine (high performance liquid chromatography) were determined. Arginase activity was inhibited by infusion of N-omega-hydroxy-nor-l-arginine (nor-NOHA, 400 mg/kg body wt, i.v.). Values are means ± s.e. Five hours of PE caused red blood cell hemolysis (15-fold increase in plasma hemoglobin) and release of arginase activity (2.7-fold increase). Plasma l-arginine concentration decreased significantly from 250 ± 20.6 to 118 ± 6.0 μmol/L (Control vs. PE) and estimated pulmonary vascular resistance increased 3-fold. Treatment with nor-NOHA prevented the depletion of plasma l-arginine (229 ± 15 μmol/L) and reduced the rise in pulmonary vascular resistance by 40%. In conclusion, experimental PE causes hemolysis, release of arginase activity, depletion of plasma l-arginine and increased estimated pulmonary vascular resistance. Inhibition of arginase activity preserves plasma l-arginine levels and improves estimated resistance, suggesting that the release of arginase during hemolysis contributes to the rise in estimated pulmonary resistance during experimental PE.

Proteomics of microparticles after experimental pulmonary embolism

IntroductionMicroparticles (MPs) are small fragments of apoptotic or activated cells that may contribute to pathological processes in cardiovascular diseases. In studies of MPs in clinical cohorts, it is unclear if observed changes in MP composition are a cause or a result of the cardiovascular disease being studied. The present studies employed a well-characterized rat model of experimental pulmonary embolism (PE) to determine if there were changes in MP characteristics as a result of pulmonary vascular occlusion. MethodsPE was produced by infusing 25μm polystyrene microspheres into the jugular vein of anesthetized rats. MPs were isolated by differential centrifugation of arterial blood 18hr after PE. Proteins were separated by 1D gel electrophoresis and identified from tryptic digests by ultraperformance liquid chromatography (UPLC) coupled with tandem mass spectrometry. Statistical analysis was conducted using the Power Law Global Error Model (PLGEM). Changes in two proteins were confirmed by Western blot. ResultsExperimental PE produced pulmonary hypertension, mild systemic hypotension, hypoxia, hypercapnia and lactic acidosis. MPs showed significant elevation in proteins involved in clotting (fibronectin precursor, fibrinogen alpha, beta and gamma and von Willebrand factor) and several macroglobulin proteins, such as alpha-2-macroglobulin precursor compared with vehicle-treated control rats. Consistent with recent observations of hemolysis in PE, haptoglobin precursor protein, a major protein of hemoglobin clearance, decreased significantly in the PE animals. Plasma d-Dimer concentrations were significantly elevated, indicating that experimental PE produced a pro-coagulant state. ConclusionsThese findings suggest that experimental PE produced significant, changes in MP characteristics to a prothrombotic phenotype.

Up-regulation of arginase II contributes to pulmonary vascular endothelial cell dysfunction during experimental pulmonary embolism

Pulmonary embolism (PE) causes pulmonary hypertension by mechanical obstruction and constriction of non-obstructed vasculature. We tested if experimental PE impairs pulmonary vascular endothelium-dependent dilation via activation of arginase II. Experimental PE was induced in male Sprague–Dawley rats by infusing 25 μm microspheres in the right jugular vein, producing moderate pulmonary hypertension. Shams received vehicle injection. Pulmonary arterial rings were isolated after 18 h and isometric tensions were determined. Dilations were induced with acetylcholine, calcium ionophore A23187 or nitroglycerin (NTG) in pre-contracted rings (phenylephrine). Protein expression was assessed by Western blot and immunohistochemistry. Arginase activity was inhibited by intravenous infusion of N w-hydroxy-nor- l-arginine (nor-NOHA). l-Arginine supplementation was also given. Endothelium-dependent dilation responses were significantly reduced in PE vs. vehicle-treated animals (ACh: 50 ± 9% vs. 93 ± 3%; A23187: 19 ± 7% vs. 85 ± 7%, p < 0.05), while endothelium-independent dilations (NTG) were unchanged. Endothelial nitric oxide synthase (eNOS) protein content was unchanged by PE. Expression of arginase II increased 4.5-fold and immunohistochemistry revealed increased arginase II staining. Nor-NOHA treatment and l-arginine supplementation significantly improved pulmonary artery ring endothelium-dependent dilation in PE (ACh: 58 ± 6% PE, 88 ± 6% PE + nor-NOHA, 84 ± 4% PE + l-arginine). Experimental PE impairs endothelium-dependent pulmonary artery dilation, while endothelium-independent dilation remains unchanged. The data support the conclusion that up-regulation of arginase II protein expression contributes to pulmonary artery endothelial dysfunction in this model of experimental PE.

Imaging Thromboembolism with 99mtc Labelled Chimeric Monoclonal Anti-Platelet Glycoprotein Iiia Antibody Fragment chSZ21F(ab)2: Evaluation In Beagle Canines

AbstractAbstract 4387 PurposeA mouse/human chimeric monoclonal antibody F(ab)2 fragment, with high affinity for platelet glycoprotein IIIa was labelled with99mTc (99mTc-chSZ21F(ab)2), and evaluated in canine to image experimental venous thromboembolism (deep vein thrombosis [DVT]) and pulmonary embolism (PE). MethodsADP-stimulated platelet aggregation was performed to determine the affinity and specificity of chSZ21F(ab)2 to the GPIIb/IIIa receptor on plates of human or canine. ChSZ21F(ab)2 was modified with 2-iminotholane and incubated with 99mTc-glucoheptonate for imaging of 24-h-old DVT and PE in a canine model. Animals were imaged for up to 3 h after injection, heparinized, and sacrificed. Lungs and other tissues, including blood and normal lungs, were harvested and, concurrently, 99mTc was counted for determination of target to tissue ratios and the percentage of injected dose per gram of tissue. ResultsThe concentration of chSZ21F(ab)2 required to inhibit 50% of platelet aggregation (IC50) in platelet-rich plasma was 11.6±7.9 nM for human and 24.9±18.8 nM for canine. In vivo, focal uptake was observed in planar images as early as 30 min (DVT), and 60 min (PE) after injection. Lesion uptake of 99mTc-chSZ21F(ab)2 at 3 h after injection was calculated in terms of percentage injected dose per gram (%ID/g) of tissue and averaged 0.076%ID/g PE and 0.083%ID/g DVT. Lesion-to-background ratios averaged 12.8 (PE-to-lung), 7.2 (DVT-to-blood), and 117.0(DVT-to-muscle). Conclusion99mTc-chSZ21F(ab)2 with its modest affinity and high DVT and PE uptake is a promising agent for imaging vascular thrombosis. Disclosures:No relevant conflicts of interest to declare.

Localization of increased lung vascular permeability in experimental pulmonary embolism and the role of PGI2

Localization of increased lung vascular permeability in experimental pulmonary embolism and the role of PGI2