Inhaled nitric oxide to control platelet hyper-reactivity in patients with acute submassive pulmonary embolism.

BACKGROUND: We test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE). METHODS: Participants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca++] without and with pharmacological soluble guanylate (sGC) modulation. RESULTS: Participants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca++] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls. CONCLUSIONS: In patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca++], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target.

Differential effect of mild and severe pulmonary embolism on the rat lung transcriptome.

BACKGROUND: Pulmonary thromboembolism (PTE) is a common diagnosis and a leading cause of cardiovascular morbidity and mortality. A growing literature has associated PE with systemic inflammation, and global hyper-coagulability, which contribute to lung remodeling and clot recurrence. The source and mechanism of inflammation remains unstudied. In humans, inhibition of cholesterol synthesis with statins decreases biomarkers of inflammation. We test the differential effect of pulmonary vascular occlusion during mild and severe pulmonary embolism on the lung transcriptome. METHODS: Experimental PE was induced in adult male rats by injection of 25 micron polystyrene microspheres into the jugular vein. The effect of Mild PE, (2-h right ventricular systolic pressure [RVSP] normal, 18-h RVSP 44 mmHg) and Severe PE (2-h RVSP > 50 mmHg; 18-h RVSP 44 mmHg) on lungs was assessed by measuring transcriptome-wide changes in gene expression by DNA microarrays. RESULTS: Severe PE was associated with a large change in lung gene expression and in the expression of KEGG pathways and other gene functional annotation groups. Mild PE was also associated with a large number of significant changes in gene expression and in the expression of KEGG pathways and gene functional annotation groups, even after only 2 h of PE. Up-regulated pathways included increased adipocytokine, chemokine and cytokine signaling as well as cholesterol synthesis. CONCLUSIONS: Mild PE without acute pulmonary hypertension (PH) increased lung gene expression of inflammatory pathways, including increased cholesterol synthesis. These data indicate that even mild persistent pulmonary vascular occlusion is capable of inciting an inflammatory response from the lung. These data imply the detrimental effect of unresolved pulmonary obstruction from PE.

A novel nontoxic inhibitor of the activation of NADPH oxidase reduces reactive oxygen species production in mouse lung.

1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02-0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67-87% of the injected dose for i.t. and 23-42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24-72 hours. Mice treated with MJ33 at 12.5-25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation.

Modulation of soluble guanylate cyclase ameliorates pulmonary hypertension in a rat model of chronic thromboembolic pulmonary hypertension by stimulating angiogenesis.

Acute pulmonary embolism (PE) does not always resolve after treatment and can progress to chronic thromboembolic disease (CTED) or the more severe chronic thromboembolic pulmonary hypertension (CTEPH). The mechanisms surrounding the likelihood of PE resolution or progress to CTED/CTEPH remain largely unknown. We have developed a rat model of CTEPH that closely resembles the human disease in terms of hemodynamics and cardiac manifestations. Embolization of rats with polystyrene microspheres followed by suppression of angiogenesis with the inhibitor of vascular endothelial growth factor receptor 2 (VEGF-R2) SU5416 results in transient, acute pulmonary hypertension that progresses into chronic PE with PH with sustained right ventricular systolic pressures exceeding 70 mmHg (chronic pulmonary embolism [CPE] model). This model is similar to the widely utilized hypoxia/SU5416 model with the exception that the "first hit" is PE. Rats with CPE have impaired right heart function characterized by reduced VO2 Max, reduced cardiac output, and increased Fulton index. None of these metrics are adversely affected by PE alone. Contrast-mediated CT imaging of lungs from rats with PE minus SU5416 show large increases in pulmonary vascular volume, presumably due to an angiogenic response to acute PE/PH. Co-treatment with SU5416 suppresses angiogenesis and produces the CTEPH-like phenotype. We report here that treatment of CPE rats with agonists for soluble guanylate cyclase, a source of cGMP which is in turn a signal for angiogenesis, markedly increases angiogenesis in lungs, and ameliorates the cardiac deficiencies in the CPE model. These results have implications for future development of therapies for human CTEPH.

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.

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."

Role of inflammation in right ventricular damage and repair following experimental pulmonary embolism in rats.

Right ventricular (RV) dysfunction is associated with poor clinical outcome following pulmonary embolism (PE). Previous studies in our laboratory show that influx of neutrophils contributes to acute RV damage seen in an 18 h rat model of PE. The present study describes the further progression of inflammation over 6 weeks and compares the neutrophil and monocyte responses. The RV outflow tract became white in colour by day 1 with influx of neutrophils (tissue myeloperoxidase activity increased 17-fold) and mononuclear cells with characteristics of M1 phenotype (high in Ccl20, Cxcl10, CcR2, MHCII, DNA microarray analysis). Matrix metalloproteinase activities were increased and tissue was thinned to produce a translucent appearance in weeks 1 through 6 in 40% of hearts. RV contractile function was significantly reduced at 6 weeks of PE. In this later phase, there was accumulation of myofibroblasts, the presence of mononuclear cells with M2 characteristics (high in scavenger mannose receptors, macrophage galactose lectin 1, PDGFR1, PDGFRbeta), enrichment of the subendocardial region of the RV outflow tract with neovesels (alpha-smooth muscle immunohistochemistry) and deposition of collagen fibres (picrosirius red staining) beginning scar formation. Thus, while neutrophil response is associated with the early, acute inflammatory events, macrophage cells continue to be present during the proliferative phase and initial deposition of collagen in this model, changing from the M1 to the M2 phenotype. This suggests that the macrophage cell response is biphasic.

Angiotensin II induces neutrophil accumulation in vivo through generation and release of CXC chemokines.

BACKGROUND: Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders in which prominent neutrophil accumulation occurs. Ang II can be generated intravascularly by the renin-angiotensin system or extravascularly by mast cell chymase. In this study, we characterized the ability of Ang II to induce neutrophil accumulation. METHODS AND RESULTS: Intraperitoneal administration of Ang II (1 nmol/L) induced significant neutrophil recruitment within 4 hours (13.3+/-2.3x10(6) neutrophils per rat versus 0.7+/-0.5x10(6) in control animals), which disappeared by 24 hours. Maximal levels of CXC chemokines were detected 1 hour after Ang II injection (577+/-224 pmol/L cytokine-inducible neutrophil chemoattractant [CINC]/keratinocyte-derived chemokine [KC] versus 5+/-3, and 281+/-120 pmol/L macrophage inflammatory protein [MIP-2] versus 14+/-6). Intravital microscopy within the rat mesenteric microcirculation showed that the short-term (30 to 60 minutes) leukocyte-endothelial cell interactions induced by Ang II were attenuated by an anti-rat CINC/KC antibody and nearly abolished by the CXCR2 antagonist SB-517785-M. In human umbilical vein endothelial cells (HUVECs) or human pulmonary artery media in culture, Ang II induced interleukin (IL)-8 mRNA expression at 1, 4, and 24 hours and the release of IL-8 at 4 hours through interaction with Ang II type 1 receptors. When HUVECs were pretreated with IL-1 for 24 hours to promote IL-8 storage in Weibel-Palade bodies, the Ang II-induced IL-8 release was more rapid and of greater magnitude. CONCLUSIONS: Ang II provokes rapid neutrophil recruitment, mediated through the release of CXC chemokines such as CINC/KC and MIP-2 in rats and IL-8 in humans, and may contribute to the infiltration of neutrophils observed in acute myocardial infarction.

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.

Contrasting roles for CXCR2 during experimental colitis.

Neutrophil recruitment into the colon is believed to play a crucial pathogenic role in the progression of clinical and experimental inflammatory bowel diseases (IBDs). The chemokine receptor CXCR2 is highly expressed on neutrophils, and promotes neutrophil recruitment in several inflammatory diseases. The present study determined the biological role of CXCR2 during trinitrobenzene sulfonic acid (TNBS)-induced colitis in the rat by assessing effects of CXCR2 antibody neutralization on neutrophil accumulation during the early (8 h) and late phase (day 7) of TNBS-induced colitis. CXCR2 expression was elevated (>3-fold above control) within 8 h and remained elevated to day 7 of colitis induction, in parallel with significant increases in neutrophil infiltration. Treatment of colitic rats with a single dose of CXCR2 neutralizing antibody significantly reduced colonic neutrophil accumulation during the early (8 h) phase of TNBS-induced colitis. However, chronic administration of CXCR2 antibody did not reduce colonic neutrophil accumulation during the late phase (day 7) of TNBS-induced colitis. In summary, the present findings suggest a functional role for CXCR2 in initiating neutrophil recruitment during the early phase of TNBS-induced acute colitis, and demonstrate that: early colonic neutrophil accumulation is CXCR2 dependent and the late phase colonic neutrophil accumulation is CXCR2 independent.

Chemokines accumulate in the lungs of rats with severe pulmonary embolism induced by polystyrene microspheres.

Pulmonary thromboembolism (PEm) is a serious and life threatening disease and the most common cause of acute pulmonary vascular occlusion. Even following successful treatment of PEm, many patients experience long-term disability due to diminished heart and lung function. Considerable damage to the lungs presumably occurs due to reperfusion injury following anti-occlusive treatments for PEm and the resulting chronic inflammatory state in the lung vasculature. We have used a rat model of irreversible PEm to ask whether pulmonary vascular occlusion in the absence of reperfusion is itself sufficient to induce an inflammatory response in lungs. By adjusting the severity of the vascular occlusion, we were able to generate hypertensive and nonhypertensive PEm, and then examine lung tissue for expression of CXC and C-C chemokine genes and bronchoalveolar lavage (BAL) fluid for the presence of chemokine proteins. Hypertensive and nonhypertensive PEm resulted in increased expression of both CXC and C-C chemokines genes in lung tissues. Hypertensive PEm was also associated with a 50-100-fold increase in protein content in lung BAL fluid, which included the CXC chemokines cytokine-induced neutrophil chemoattractant and macrophage-inflammatory protein 2. The presence of chemokines in BALs was reflected by a potent neutrophil chemotactic activity in in vitro chemotaxis assays. Abs to cytokine-induced neutrophil chemoattractant blocked the in vitro neutrophil chemotactic activity of BAL by 44%. Our results indicate that the ischemia and hypertension associated with PEm are sufficient to induce expression of proinflammatory mediators such as chemokines, and establish a proinflammatory environment in the ischemic lung even before reperfusion.