Person:

Otterbein, Leo

Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery.

Purpose Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC). Methods: To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10–100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H2DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue. Results: ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25±2% rotenone vs. 14±1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17±4% rotenone vs. 55±3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20±1% ODQ+ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255±327 RGC/mm2 vs. ALF186+IRI 2036±83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186+IRI 2036±83 RGC/mm2 vs. NS-2028+ALF186+IRI 1263±170, p<0.05). Conclusions: The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain.

Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. Methodology/Principal Findings: We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca2+]i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca2+]i in mouse sickle erythrocytes did not require KCa3.1 activity. Conclusions/Significance: The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

The articles contained in this review series exemplify the diverse applications and succinct biological relevance of this simple gas. Articles summarizing the important effects of carbon monoxide in preventing the rejection of an organ, in its neuroprotective properties in piglets, regulation of mycobacterial growth, in its anti-inflammatory effects in the gut and in its use in new and innovative modalities and avenues by which to harness adjuvant therapies are eloquently and precisely described and reviewed. Each of these reports offers but a glimpse of continued prudent and sound evidence that this simple diatomic gas offers astonishingly potent and extremely diverse biological and medicinal qualities.

Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.

Islet transplantation is the most valid approach to the treatment of type 1 diabetes. However, the function of transplanted islets is often compromised since a large number of β cells undergo apoptosis induced by stress and the immune rejection response elicited by the recipient after transplantation. Conventional treatment for islet transplantation is to administer immunosuppressive drugs to the recipient to suppress the immune rejection response mounted against transplanted islets. Induction of protective genes in the recipient (e.g., heme oxygenase-1 (HO-1), A20/tumor necrosis factor alpha inducible protein3 (tnfaip3), biliverdin reductase (BVR), Bcl2, and others) or administration of one or more of the products of HO-1 to the donor, the islets themselves, and/or the recipient offers an alternative or synergistic approach to improve islet graft survival and function. In this perspective, we summarize studies describing the protective effects of these genes on islet survival and function in rodent allogeneic and xenogeneic transplantation models and the prevention of onset of diabetes, with emphasis on HO-1, A20, and BVR. Such approaches are also appealing to islet autotransplantation in patients with chronic pancreatitis after total pancreatectomy, a procedure that currently only leads to 1/3 of transplanted patients being diabetes-free.

Ischemia reperfusion injury (IRI) in organ transplantation remains a serious and unsolved problem. Organs that undergo significant damage during IRI, function less well immediately after reperfusion and tend to have more problems at later times when rejection can occur. Biliverdin has emerged as an agent that potently suppress IRI in rodent models. Since the use of biliverdin is being developed as a potential therapeutic modality for humans, we tested the efficacy for its effects on IRI of the liver in swine, an accepted and relevant pre-clinical animal model. Administration of biliverdin resulted in rapid appearance of bilirubin in the serum and significantly suppressed IRI-induced liver dysfunction as measured by multiple parameters including urea and ammonia clearance, neutrophil infiltration and tissue histopathology including hepatocyte cell death. Taken together, our findings, in a large animal model, provide strong support for the continued evaluation of biliverdin as a potential therapeutic in the clinical setting of transplantation of the liver and perhaps other organs.

Rationale: Emphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders. Objectives: To discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers. Methods: Replicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays. Measurements and Main Results Circulating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7–5.7, p = 0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7–13.3, p = 0.002), and increased circulating bone metabolites (p = 0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002) and IFN-gamma production (p = 0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005) and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively). Conclusions: Humoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance for the pathogenesis of smoking-associated diseases, and development of biomarker immunoassays and/or novel treatments for these disorders.

We hypothesized that tumor-associated macrophages (TAMs) are controlled by the diffusible gas carbon monoxide (CO). We demonstrate that induction of apoptosis in lung tumors treated with low doses of CO is associated with increased CD86 expression and activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (Erk) 1/2 pathway in tumor microenvironment. Presence of CD86-positive cells was required for the anti-tumoral effects of CO in established A549 xenografts. We show that the effects of CO on tumor stroma and reprogramming of macrophages towards the anti-tumoral phenotype is mediated by reactive oxygen species (ROS)-dependent activation of MAPK/Erk1/2-c-myc pathway as well as Notch 1-dependent negative feedback on the metabolic enzyme heme oxygenase-1 (HO-1). We find a similar negative correlation between HO-1 and active MAPK-Erk1/2 levels in human lung cancer specimens. In summary, we describe novel non-cell autonomous mechanisms by which the diffusible gas CO dictates changes in the tumor microenvironment through the modulation of macrophages.

Cystatin C is believed to prevent tumor progression by inhibiting the activities of a family of lysosomal cysteine proteases. However, little is known about the precise mechanism of cystatin C function in prostate cancer. In the present study, we examined the expression of cystatin C and its association with matrix metalloproteinases 2 (MMP2) and androgen receptor (AR) in a tissue microarray comparing benign and malignant specimens from 448 patients who underwent radical prostatectomy for localized prostate cancer. Cystatin C expression was significantly lower in cancer specimens than in benign tissues (p<0.001) and there was a statistically significant inverse correlation between expression of cystatin C and MMP2 (rs2 = −0.056, p = 0.05). There was a clear trend that patients with decreased level of cystatin C had lower overall survival. Targeted inhibition of cystatin C using specific siRNA resulted in an increased invasiveness of PC3 cells, whereas induction of cystatin C overexpression greatly reduced invasion rate of PC3 in vitro. The effect of cystatin C on modulating the PC3 cell invasion was provoked by Erk2 inhibitor that specifically inhibited MAPK/Erk2 activity. This suggests that cystatin C may mediate tumor cell invasion by modulating the activity of MAPK/Erk cascades. Consistent with our immunohistochemical findings that patients with low expression of cystatin C and high expression of androgen receptor (AR) tend to have worse overall survival than patients with high expression of cystatin C and high AR expression, induced overexpression of AR in PC3 cells expressing cystatin C siRNA greatly enhanced the invasiveness of PC3 cells. This suggests that there may be a crosstalk between cystatin C and AR-mediated pathways. Our study uncovers a novel role for cystatin C and its associated cellular pathways in prostate cancer invasion and metastasis.