A soluble guanylate cyclase stimulator, BAY 41-8543, preserves right ventricular function in experimental pulmonary embolism.

Pulmonary embolism (PE) increases pulmonary vascular resistance, causing right ventricular (RV) dysfunction, and poor clinical outcome. Present studies test if the soluble guanylate cyclase stimulator BAY 41-8543 reduces pulmonary vascular resistance and protects RV function. Experimental PE was induced in anesthetized, male Sprague-Dawley rats by infusing 25 µm polystyrene microspheres (1.95 million/100 g body wt, right jugular vein) producing moderate PE. Pulmonary artery vascular resistance, estimated as RVPSP/CO, increased 3-fold after 5 h of PE. Treatment with BAY 41-8543 (50 µg/kg, I.V.; given at the time of PE induction) normalized this index by reducing RVPSP and markedly increasing CO, via preservation of heart rate and stroke volume. Ex vivo RV heart function showed minimal changes at 5 h of PE, but decreased significantly after 18 h of PE, including peak systolic pressure (PSP, Control 39 ± 1 mmHg vs. 19 ± 3 PE), +dP/dt (1192 ± 93 mmHg/s vs. 444 ± 64) and -dP/dt (-576 ± 60 mmHg/s vs. -278 ± 40). BAY 41-8543 significantly improved all three indices of RV heart function (PSP 35 ± 3.5, +dP/dt 1129 ± 100, -dP/dt -568 ± 87). Experimental PE produced increased PVR and RV dysfunction, which were ameliorated by treatment with BAY 41-8543. Thus, there is vasodilator reserve in this model of experimental PE that can be exploited to reduce the stress upon the heart and preserve RV contractile function.

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.

INTRODUCTION: Microparticles (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. METHODS: PE was produced by infusing 25 µm polystyrene microspheres into the jugular vein of anesthetized rats. MPs were isolated by differential centrifugation of arterial blood 18 hr 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. RESULTS: Experimental 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. CONCLUSIONS: These findings suggest that experimental PE produced significant, changes in MP characteristics to a prothrombotic phenotype.

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

OBJECTIVES: 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. DESIGN: Controlled animal study. SETTING: Medical center research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: 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. MEASUREMENTS AND MAIN RESULTS: 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%. CONCLUSIONS: 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.

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.

Development and comparison of a minimally-invasive model of autologous clot pulmonary embolism in Sprague-Dawley and Copenhagen rats.

BACKGROUND: Experimental models of pulmonary embolism (PE) that produce pulmonary hypertension (PH) employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. OBJECTIVE: Test if moderate-severity autologous PE in Sprague-Dawley (SD) and Copenhagen (Cop) rats can produce persistent PH. METHODS: blood was withdrawn from the jugular vein, treated with thrombin-Ca++ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days. RESULTS: Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours) and 97% (5 days) in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days). Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation. CONCLUSIONS: Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion.

Transcriptional changes in right ventricular tissues are enriched in the outflow tract compared with the apex during chronic pulmonary embolism in rats.

Moderate to severe pulmonary embolism (PE) can cause pulmonary arterial hypertension and right ventricular (RV) heart damage. Previous studies from our laboratory indicate that the basal outflow tract of the RV is injured and has acute inflammation followed by tissue remodeling while the apex appears normal. The present studies examine transcription responses to chronic PE in RV apex and outflow tracts using DNA microarrays to identify transcription responses by region. Changes predominated in the RV outflow tract (8,575 genes showed >/=1.5-fold expression change). Gene ontology and KEGG analyses indicated a significant decrease in genes involved in cellular respiration and energy metabolism and increases in inflammatory cell adhesion molecules and extracellular matrix proteins. Signal pathways for wound healing such as fibroblast growth factor, collagen synthesis, and CCN proteins (named for the first three members of the family: cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene) were strongly upregulated. In comparison, few genes (422) showed significant change in the RV apex tissue. Apex-selective genes included two genes affecting metabolism and a stretch-sensitive transcription factor (ankyrin repeat domain 1). We conclude that the RV outflow tract is subject to strong proinflammatory and profibrotic remodeling transcriptional responses in chronic PE. Severe loss of genes involved in cellular respiration is consistent with previous histology indicating a shift in cell types present within the outflow tract tissue away from highly energy-dependant cardiomyocytes to less metabolically active cells during remodeling. The apex region of the RV had few compensating adaptations.

Proinflammatory events in right ventricular damage during pulmonary embolism: effects of treatment with ketorolac in rats.

Right ventricular (RV) damage contributes to poor clinical outcome after pulmonary embolism (PE). Our studies show that neutrophils contribute to RV dysfunction in rat PE. Present studies examine effects of the nonsteroidal anti-inflammatory drug, ketorolac, upon RV inflammation and dysfunction. RV inflammatory gene expression significantly increased 6 and 18 hours after PE [cytokine-induced neutrophil chemoattractant-1 (CINC-1) 18-fold and 24-fold; cyclooxygenase-2 21-fold and 32-fold]. Eighteen hours after PE, there was significant upregulation of adhesion molecules (selectin E 18-fold; intercellular adhesion molecule 1 14-fold), influx of neutrophils (myeloperoxidase activity 21-fold), depressed RV function (RV peak systolic pressure = 24 +/- 3 vs. 40 +/- 1 mm Hg; maximum rate of pressure development = 444 +/- 79 vs. 1533 +/- 146; maximum rate of pressure decrease = -357 +/- 50 vs. -651 +/- 44), and release of cardiac troponin I (7.8 +/- 1.9 ng/mL) compared with vehicle. Ketorolac (10 mg/kg, intraperitoneally) significantly reduced expression of CINC-1, cyclooxygenase-2, selectin E, and intercellular adhesion molecule 1, lowered neutrophil influx, improved RV function (RV peak systolic pressure was 34 +/- 3 mm Hg; maximum rate of pressure development = 1288 +/- 146; maximum rate of pressure decrease = -611 +/- 92), and marginally reduced cardiac troponin I release (P < 0.07) compared with PE alone. Ketorolac reduced CINC-1 stimulated chemotaxis of isolated neutrophils. PE converted cardiac tissue into a proinflammatory phenotype. Ketorolac reduced RV inflammatory genes, reduced neutrophil influx, and improved RV function in rat PE.

Transcriptional profile of right ventricular tissue during acute pulmonary embolism in rats.

Acute pulmonary embolism (PE) is the third leading cause of cardiovascular death in the United States. Moderate to severe PE can cause pulmonary arterial hypertension (PH) with resultant right ventricular (RV) heart damage. The mechanisms leading to RV failure after PE are not well defined, although it is becoming clear that PH-induced inflammatory responses are involved. We previously demonstrated profound neutrophil-mediated inflammation and RV dysfunction during PE that was associated with increased expression of several chemokine genes. However, a complete assessment of transcriptional changes in RVs during PE is still lacking. We have now used DNA microarrays to assess the alterations in gene expression in RV tissue during acute PE/PH in rats. Key results were confirmed with real-time RT-PCR. Nine CC-chemokine genes (CCL-2, -3, -4, -6, -7, -9, -17, -20, -27), five CXC-chemokine genes (CXCL-1, -2, -9, -10, -16), and the receptors CCR1 and CXCR4 were upregulated after 18 h of moderate PE, while one C-chemokine (XCL-1) and one CXC-chemokine (CXCL-12) were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated increased expression of many inflammatory genes. There was also a major shift in the expression of components of metabolic pathways, including downregulation of fatty acid transporters and oxidative enzymes, a change in glucose transporters, and upregulation of stretch-sensing and hypoxia-inducible transcription factors. This pattern suggests an extensive shift in cardiac physiology favoring the expression of the "fetal gene program."

Inhibition of CINC-1 decreases right ventricular damage caused by experimental pulmonary embolism in rats.

Right ventricular (RV) dysfunction is a strong risk factor for poor clinical outcome following pulmonary embolism (PE), the third most prevalent cardiovascular disease. Previous studies in our laboratory demonstrated that RV failure during PE is mediated, in part, by neutrophil-dependant cardiac inflammation. In this study we use DNA microarray analysis of gene expression to demonstrate that PE results in increased expression of the CXC chemokines CINC-1 and CINC-2 between 6 and 18 h after the start of PE in a rat model of PE. Neutrophils accumulate in RV tissue by 18 h, and this inflammation is associated with decreased right heart function. Treatment of rats with Abs to CINC-1 significantly suppressed neutrophil accumulation in RVs during PE (52% reduction in tissue myeloperoxidase) and ameliorated RV failure. In addition, plasma concentration of cardiac troponin I, an established diagnostic marker for cardiac damage, was reduced by 90%. These results suggest that selective anti-inflammatory therapies targeted at neutrophil chemoattractants will reduce cardiac inflammation and reduce RV damage in the setting of PE.

Cardiac inflammation contributes to right ventricular dysfunction following experimental pulmonary embolism in rats.

Acute right ventricular (RV) failure following pulmonary embolism (PE) is a strong predictor of poor clinical outcome. Present studies test for an association between RV failure from experimental PE, inflammation, and upregulated chemokine expression. Additional experiments test if neutrophil influx contributes to RV dysfunction. PE was induced in male rats by infusing 24 microm microspheres (right jugular vein) producing mild hypertension (1.3 million beads/100 g, PE1.3), or moderately severe hypertension (2.0 million beads/100 g, PE2.0). Additional rats served as vehicle sham (0.01% Tween 20, Veh). In vivo RV peak systolic pressures (RVPSP) increased significantly, and then declined following PE2.0 (51 +/- 1 mm Hg 2 h; 49 +/- 1, 6 h; 44 +/- 1, 18 h). RV generated pressure of isolated, perfused hearts was significantly reduced in PE2.0 compared with PE1.3 or Veh. MCP-1 protein (ELISA) was elevated 21-fold and myeloperoxidase activity 95-fold in RV of PE2.0 compared with Veh or PE1.3. CINC-1, CINC-2, MIP-2, MCP-1, and MIP-1alpha mRNA also increased in RV of PE2.0. Histological analysis revealed massive accumulation of neutrophils (selective esterase stain) and monocyte/macrophages (CD68, ED-1) in RV of PE2.0 hearts in regions of myocyte damage. Electron microscopy showed myocyte necrosis and phagocytosis by inflammatory cells. LV function was normal and did not show increased inflammation after PE2.0. Treatment with anti-PMN antibody reduced RV MPO activity and prevented RV dysfunction. Conclusions-PE with moderately severe pulmonary hypertension (PE2.0) resulted in selective RV dysfunction, which was associated with increased chemokine expression, and infiltration of both neutrophils and monocyte/macrophages, indicating that a robust immune response occurred with RV damage following experimental PE. Experimental agranulocytosis reduced RV, suggesting that neutrophil influx contributed to RV damage.