Sean Alber

Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells

BACKGROUND Preclinical atherosclerosis is associated with increased endothelial cell (EC) expression of leukocyte adhesion molecules (LAMs), which mediate monocyte adhesion during atherogenesis. Identification of cell-surface LAMs may uniquely allow assessment of endothelial function, but there are no in vivo methods for detecting LAMs. We tested a new microbubble designed to bind to and allow specific ultrasound detection of intercellular adhesion molecule-1 (ICAM-1). METHODS AND RESULTS A perfluorobutane gas-filled lipid-derived microsphere with monoclonal antibody to ICAM-1 covalently bound to the bubble shell was synthesized. Bubbles with either nonspecific IgG or no protein on the shell were synthesized as controls. Coverslips of cultured human coronary artery ECs were placed in a parallel-plate perfusion chamber and exposed to 1 of the 3 microbubble species, followed by perfusion with culture medium. Experiments were performed with either normal or interleukin-1beta-activated ECs overexpressing ICAM-1, and bubble adherence was quantified with epifluorescent videomicroscopy. There was limited adherence of control bubbles to normal or activated ECs, whereas a 40-fold increase in adhesion occurred when anti-ICAM-1-conjugated bubbles were exposed to activated ECs compared with normal ECs (8.1+/-3.5 versus 0.21+/-0.09 bubbles per cell, respectively, P<0.001). Although diminished, this difference persisted even after perfusion at higher wall shear rates. CONCLUSIONS A gas-filled microbubble with anti-ICAM-1 antibody on its shell specifically binds to activated ECs overexpressing ICAM-1. Diagnostic ultrasound in conjunction with targeted contrast agents has the unique potential to characterize cell phenotype in vivo.

IL-18-induced CD83+CCR7+ NK helper cells

In addition to their cytotoxic activities, natural killer (NK) cells can have immunoregulatory functions. We describe a distinct “helper” differentiation pathway of human CD56+CD3− NK cells into CD56+/CD83+/CCR7+/CD25+ cells that display high migratory responsiveness to lymph node (LN)–associated chemokines, high ability to produce interferon-γ upon exposure to dendritic cell (DC)- or T helper (Th) cell–related signals, and pronounced abilities to promote interleukin (IL)-12p70 production in DCs and the development of Th1 responses. This helper pathway of NK cell differentiation, which is not associated with any enhancement of cytolytic activity, is induced by IL-18, but not other NK cell–activating factors. It is blocked by prostaglandin (PG)E2, a factor that induces a similar CD83+/CCR7+/CD25+ LN-homing phenotype in maturing DCs. The current data demonstrate independent regulation of the “helper” versus “effector” pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE2.

Carbon Monoxide Induces Cytoprotection in Rat Orthotopic Lung Transplantation via Anti-Inflammatory and Anti-Apoptotic Effects

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

Carbon Monoxide Suppresses Bleomycin-Induced Lung Fibrosis

Idiopathic pulmonary fibrosis is an incurable fibrosing disorder that progresses relentlessly to respiratory failure. We hypothesized that a product of heme oxygenase activity, carbon monoxide (CO), may have anti-fibrotic effects. To test this hypothesis, mice treated with intratracheal bleomycin were exposed to low-concentration inhaled CO or ambient air. Lungs of mice treated with CO had significantly lower hydroxyproline accumulation than controls. Fibroblast proliferation, thought to play a central role in the progression of fibrosis, was suppressed by in vitro exposure to CO. CO caused increased cellular levels of p21(Cip1) and decreased levels of cyclins A and D. This effect was independent of the observed suppression of MAPKs phosphorylation by CO but was dependent on increased cGMP levels. Further, CO-exposed cells elaborated significantly less fibronectin and collagen-1 than control cells. This same effect was seen in vivo. Suppression of collagen-1 production did not depend on MAPK or guanylate cyclase signaling pathways but did depend on the transcriptional regulator Id1. Taken together, these data suggest that CO exerts an anti-fibrotic effect in the lung, and this effect may be due to suppression of fibroblast proliferation and/or suppression of matrix deposition by fibroblasts.

Sunitinib facilitates the activation and recruitment of therapeutic anti-tumor immunity in concert with specific vaccination

The multikinase inhibitor sunitinib malate (SUT) has been reported to reduce levels of myeloid suppressor cells and Treg cells in cancer patients, hypothetically diminishing intrinsic impediments for active immunization against tumor‐associated antigens in such individuals. The goal of this study was to identify longitudinal immune molecular and cellular changes associated with tumor regression and disease‐free status after the treatment of established day 7 s.c. MO5 (B16.OVA) melanomas with SUT alone (1 mg/day via oral gavage for 7 days), vaccination using ovalbumin (OVA) peptide‐pulsed dendritic cell [vaccine (VAC)] alone, or the combination of SUT and VAC (SUT/VAC). We observed superior anti‐tumor efficacy for SUT/VAC combination approaches, particularly when SUT was applied at the time of the initial vaccination or the VAC boost. Treatment effectiveness was associated with the acute loss of (and/or failure to recruit) cells bearing myeloid‐derived suppressor cells or Treg phenotypes within the tumor microenvironment (TME) and the corollary, prolonged enhancement of Type‐1 anti‐OVA CD8+ T cell responses in the tumor‐draining lymph node and the TME. Enhanced Type‐1 T cell infiltration of tumors was associated with treatment‐induced expression of vascular cell adhesion molecule‐1 (VCAM‐1) and CXCR3 ligand chemokines in vascular/peri‐vascular cells within the TME, with SUT/VAC therapy benefits conditionally negated upon adminsitration of CXCR3 or VCAM‐1 blocking antibodies. These data support the ability of a short 7 day course of SUT to (re)condition the TME to become more receptive to the recruitment and prolonged therapeutic action of (VAC‐induced) anti‐tumor Tc1 cells.

Downregulation of Endothelin-1 by Farnesoid X Receptor in Vascular Endothelial Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine. FXR may play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. In this study, we report that FXR is also expressed in rat pulmonary artery endothelial cells (EC), a “nonclassical” bile acid target tissue. FXR is functional in EC, as demonstrated by induction of its target genes such as small heterodimer partner (SHP) after treatment with chenodeoxycholic acid, a FXR agonist. Interestingly, activation of FXR in EC led to downregulation of endothelin (ET)-1 expression. Reporter assays showed that activation of FXR inhibited transcriptional activation of the human ET-1 gene promoter and also repressed the activity of a heterologous promoter driven by activator protein (AP)-1 response elements. Electrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR reduced the binding activity of AP-1 transcriptional factors, suggesting that FXR may suppress ET-1 expression via negatively interfering with AP-1 signaling. These studies suggest that FXR may play a role in endothelial homeostasis and may serve as a novel molecular target for manipulating ET-1 expression in vascular EC.

Nitric Oxide and Ionizing Radiation Synergistically Promote Apoptosis and Growth Inhibition of Cancer by Activating p53

Nitric oxide (NO) is a potent tumor radiosensitizer; however, its clinical use is limited by systemic side effects. We have demonstrated previously that gene transfer of the human inducible NO synthase (iNOS) gene into tumor cells and tumors induces high-output NO production that significantly enhances tumor radioresponsiveness, with no observed side effects. Notably, iNOS gene transfer enhances tumor radioresponsiveness via apoptotic cell death. Because NO and ionizing radiation are both known to promote p53-dependent apoptosis, we hypothesized that p53 activation might be a primary mechanism for the synergy of these two genotoxic stresses. We report that NO and ionizing radiation synergistically activate p53 in colorectal cancers grown in athymic mice by augmenting phosphorylation of p53 at serine 15. The effect of NO and ionizing radiation on tumor cell apoptosis and tumor radioresponsiveness is significantly reduced in p53 knockout isogenic cancer cell lines. Furthermore, the transfer of both p53 and iNOS genes into tumor cells lacking functional p53 enhanced their radioresponsiveness more than transfer of either gene alone.

Autophagy is increased in mice after traumatic brain injury and is detectable in human brain after trauma and critical illness.

Autophagy is a homeostatic process for recycling of proteins and organelles, that increases during times of nutrient deprivation and is regulated by reactive oxygen species. We reported that autophagy can also be induced after traumatic brain injury (TBI) in mice.1 Specifically, autophagosomes and multilamellar bodies were frequently observed in cell processes and axons in injured brain regions by electron microscopy, and lipidated microtubule-associated protein light chain 3 (LC3-II), was increased after TBI vs. controls. To determine if antioxidants could reduce autophagy, separate mice were treated with the antioxidant γ-glutamylcysteinyl ethyl ester (GCEE). Treatment with GCEE preserved total antioxidant reserves, reduced LC3-II in injured brains, and improved both behavioral and histological outcome after TBI. Here we report that LC3-II and autophagosomes were detectable in brain tissue from humans after TBI. Taken together, we show that autophagy occurs after both experimental and clinical TBI, and that oxidative stress contributes to overall neuropathology after TBI in mice, at least in part by initiating or influencing autophagy. Addendum to: Lai Y, Hickey RW, Chen Y, Bayir H, Sullivan M, Chu CT, Kochanek PM, Dixon CE, Jenkins LW, Graham SH, Watkins SC, Clark RSB. Autophagy is increased after traumatic brain injury in mice and is partially inhibited by the antioxidant gamma-glutamylcysteinyl ethyl ester. J Cereb Blood Flow Metab 2007; In press.

Carbon Monoxide Protects against Ventilator-induced Lung Injury via PPAR-γ and Inhibition of Egr-1

RATIONALE Ventilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option. OBJECTIVES This study explores the mechanisms of CO-dependent protection in a mouse model of VILI. METHODS Mice were ventilated (12 ml/kg, 1-8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines. MEASUREMENTS AND MAIN RESULTS Mechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1-deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-gamma, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO. CONCLUSIONS Mechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-gamma and inhibition of Egr-1.

Ethyl pyruvate ameliorates distant organ injury in a murine model of acute necrotizing pancreatitis.

Objective:Ethyl pyruvate has been shown to be an effective anti-inflammatory agent in a variety of in vitro and in vivo model systems. Herein, we used a murine model of acute pancreatitis to compare the effects of treatment with either Ringer’s lactate solution or ethyl pyruvate solution on several physiologic and biochemical variables related to disease severity. Design:Experimental animal study. Setting:University laboratory. Subjects:C57Bl/6 mice. Interventions:Pancreatitis was induced by feeding the animals a choline-deficient diet supplemented with 0.5% ethionine for 24 hrs and then challenging the animals with seven hourly 50 μg/kg intraperitoneal injections of cerulein and a single intraperitoneal injection of Escherichia coli lipopolysaccharide (4 mg/kg). Measurements and Main Results:When mice were treated with ethyl pyruvate (40 mg/kg intraperitoneally every 6 hrs for 48 hrs) instead of Ringer’s lactate solution starting 2 hrs after the injection of lipopolysaccharide, long-term survival was improved from one of ten to six of ten (p = .057). When mice were treated with a 40 mg/kg dose of ethyl pyruvate just before the first dose of cerulein and then injected with a second 40 mg/kg dose 6 hrs later, serum concentrations of alanine aminotransferase measured 10 hrs after the first cerulein dose were significantly lower than in mice with pancreatitis treated with Ringer’s lactate solution. In this model of acute pancreatitis, the same dosing regimen for ethyl pyruvate also ameliorated bacterial translocation to mesenteric lymph nodes and leakage of fluorescein isothiocyanate-labeled albumin from blood into bronchoalveolar lavage fluid. Treatment with ethyl pyruvate decreased pancreatic expression of tumor necrosis factor and interleukin-6 messenger RNA and nuclear factor-κB DNA binding in nuclear extracts prepared from pancreatic tissue. Conclusion:Treatment with ethyl pyruvate ameliorated the local inflammatory response and decreased local and distant organ injury in a murine model of necrotizing pancreatitis.