Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1flfl), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation

Microwave and Physical Electronics

Contains reports on six research projects.Office of Scientific Research and Development (OSRD) OEMsr-26

Comparative analysis of an experimental subcellular protein localization assay and in silico prediction methods

The subcellular localization of a protein can provide important information about its function within the cell. As eukaryotic cells and particularly mammalian cells are characterized by a high degree of compartmentalization, most protein activities can be assigned to particular cellular compartments. The categorization of proteins by their subcellular localization is therefore one of the essential goals of the functional annotation of the human genome. We previously performed a subcellular localization screen of 52 proteins encoded on human chromosome 21. In the current study, we compared the experimental localization data to the in silico results generated by nine leading software packages with different prediction resolutions. The comparison revealed striking differences between the programs in the accuracy of their subcellular protein localization predictions. Our results strongly suggest that the recently developed predictors utilizing multiple prediction methods tend to provide significantly better performance over purely sequence-based or homology-based predictions

Inhaled carbon monoxide protects against the development of shock and mitochondrial injury following hemorrhage and resuscitation

Aims: Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation. Results: Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01). Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05). A dose response of CO (25-500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively. CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100-500ppm). Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation. In porcine HS/R, CO did not influence hemodynamics. However, CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets). Conclusion: These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R; however, further clinical studies are warranted

Complement C3 Deficiency Attenuates Chronic Hypoxia-Induced Pulmonary Hypertension in Mice

Background: Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension. Methodology/Principal Findings: Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3-/- hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3-/- mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3-/- mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3-/- mice. Conclusions: Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans. © 2011 Bauer et al

O-GlcNAc Modification of NFκB p65 Inhibits TNF-α-Induced Inflammatory Mediator Expression in Rat Aortic Smooth Muscle Cells

BACKGROUND: We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling. METHODOLOGY/PRINCIPAL FINDINGS: Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10(-4) M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65. CONCLUSIONS: There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury

Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients

Sickness behaviour pushed too far – the basis of the syndrome seen in severe protozoal, bacterial and viral diseases and post-trauma

Certain distinctive components of the severe systemic inflammatory syndrome are now well-recognized to be common to malaria, sepsis, viral infections, and post-trauma illness. While their connection with cytokines has been appreciated for some time, the constellation of changes that comprise the syndrome has simply been accepted as an empirical observation, with no theory to explain why they should coexist. New data on the effects of the main pro-inflammatory cytokines on the genetic control of sickness behaviour can be extended to provide a rationale for why this syndrome contains many of its accustomed components, such as reversible encephalopathy, gene silencing, dyserythropoiesis, seizures, coagulopathy, hypoalbuminaemia and hypertriglyceridaemia. It is thus proposed that the pattern of pathology that comprises much of the systemic inflammatory syndrome occurs when one of the usually advantageous roles of pro-inflammatory cytokines – generating sickness behaviour by moderately repressing genes (Dbp, Tef, Hlf, Per1, Per2 and Per3, and the nuclear receptor Rev-erbα) that control circadian rhythm – becomes excessive. Although reversible encephalopathy and gene silencing are severe events with potentially fatal consequences, they can be viewed as having survival advantages through lowering energy demand. In contrast, dyserythropoiesis, seizures, coagulopathy, hypoalbuminaemia and hypertriglyceridaemia may best be viewed as unfortunate consequences of extreme repression of these same genetic controls when the pro-inflammatory cytokines that cause sickness behaviour are produced excessively. As well as casting a new light on the previously unrationalized coexistence of these aspects of systemic inflammatory diseases, this concept is consistent with the case for a primary role for inflammatory cytokines in their pathogenesis across this range of diseases