Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP.

ABSTRACT: Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody's Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum-sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

Trogocytosis drives red blood cell antigen loss in association with antibody-mediated immune suppression.

ABSTRACT: Red blood cell (RBC) alloimmunization to paternal antigens during pregnancy can cause hemolytic disease of the fetus and newborn (HDFN). This severe and potentially fatal neonatal disorder can be prevented by the administration of polyclonal anti-D through a mechanism referred to as antibody-mediated immune suppression (AMIS). Although anti-D prophylaxis effectively prevents HDFN, a lack of mechanistic clarity has hampered its replacement with recombinant agents. The major theories behind AMIS induction in the hematologic literature have classically centered around RBC clearance; however, antigen modulation/loss has recently been proposed as a potential mechanism of AMIS. To explore the primary mechanisms of AMIS, we studied the ability of 11 different antibodies to induce AMIS, RBC clearance, antigen loss, and RBC membrane loss in the HOD (hen egg lysozyme-ovalbumin-human Duffy) murine model. Antibodies targeting different portions of the HOD molecule could induce AMIS independent of their ability to clear RBCs; however, all antibodies capable of inducing a strong AMIS effect also caused significant in vivo loss of the HOD antigen in conjunction with RBC membrane loss. In vitro studies of AMIS-inducing antibodies demonstrated simultaneous RBC antigen and membrane loss, which was mediated by macrophages. Confocal live-cell microscopy revealed that AMIS-inducing antibodies triggered RBC membrane transfer to macrophages, consistent with trogocytosis. Furthermore, anti-D itself can induce trogocytosis even at low concentrations, when phagocytosis is minimal or absent. In view of these findings, we propose trogocytosis as a mechanism of AMIS induction.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean + 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets.

Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA. However, there is currently no widely available monocyte or macrophage cell line expressing all these receptors. Common sources of monocytes for differentiation into macrophages, such as human peripheral blood monocytes and the monocytic leukemia cell line THP-1, generally lack the expression of FcγRIIIA (CD16A). Here, we utilized a lentiviral system to generate THP-1 cells stably expressing human FcγRIIIA (CD16F158). THP-1-CD16A cells treated with phorbol 12-myristate 13-acetate for 24 hours phagocytosed anti-D-opsonized human red blood cells primarily utilizing FcγRI with a lesser but significant contribution of IIIA while phagocytosis of antibody-opsonized human platelets equally utilized FcγRI and Fcγ IIIA. Despite the well-known ability of FcγRIIA to bind IgG in cell free systems, this receptor did not appear to be involved in either RBC or platelet phagocytosis. These transgenic cells may constitute a valuable tool for studying macrophage FcγR utilization and function.

Protective function of DJ-1/PARK7 in lipopolysaccharide and ventilator-induced acute lung injury.

Oxidative stress is considered one of the early underlying contributors of acute lung injury (ALI) and ventilator-induced lung injury (VILI). DJ-1, also known as PARK7, has a well-established role as an antioxidant. We have previously shown maintaining oxidative balance via the ATF3-Nrf2 axis was important in protection from ALI. Here, we exclusively characterize the role of DJ-1 in sterile LPS-induced ALI and VILI. DJ-1 protein expression was increased after LPS treatment in human epithelial and endothelial cell lines and lungs of wild-type mice. DJ-1 deficient mice exhibited greater susceptibility to LPS-induced acute lung injury as demonstrated by increased cellular infiltration, augmented levels of pulmonary cytokines, enhanced ROS levels and oxidized by-products, increased pulmonary edema and cell death. In a two-hit model of LPS and mechanical ventilation (MV), DJ-1 deficient mice displayed enhanced susceptibility to inflammation and lung injury. Collectively, these results identify DJ-1 as a negative regulator of ROS and inflammation, and suggest its expression protects from sterile lung injury driven by high oxidative stress.

Systemic angiopoietin-1/2 dysregulation following cardiopulmonary bypass in adults.

AIM: Vascular leakage following cardiopulmonary bypass contributes to morbidity. Angiopoietin-1 and -2 are biomarkers of endothelial dysfunction. Our aim was to characterize Ang-1 and -2 association with clinical characteristics and outcomes. METHODS: Observational cohort study measuring Ang-1/-2 with a panel of cytokines in adults undergoing cardiopulmonary bypass. RESULTS: Ang-2 levels increased immediately postop whereas Ang-1 levels decreased over time. No significant correlation was found with other inflammatory mediators. High correlation was found between the hospital length of stay and Ang-2 increase at 24 h (rho = 0.590; p < 0.0001). The predictors of Ang-2 increase were female gender, cross clamp time, transfusion of blood and absence of angiotensin-converting enzyme inhibitor as a pre-op medication. CONCLUSION: Angiopoietins can detect vascular leakage early and could impact patient's management to decrease length of stay after cardiac surgery.

DJ-1/PARK7 Impairs Bacterial Clearance in Sepsis.

RATIONALE: Effective and rapid bacterial clearance is a fundamental determinant of outcomes in sepsis. DJ-1 is a well-established reactive oxygen species (ROS) scavenger. OBJECTIVES: Because cellular ROS status is pivotal to inflammation and bacterial killing, we determined the role of DJ-1 in bacterial sepsis. METHODS: We used cell and murine models with gain- and loss-of-function experiments, plasma, and cells from patients with sepsis. MEASUREMENTS AND MAIN RESULTS: Stimulation of bone marrow-derived macrophages (BMMs) with endotoxin resulted in increased DJ-1 mRNA and protein expression. Cellular and mitochondrial ROS was increased in DJ-1-deficient (-/-) BMMs compared with wild-type. In a clinically relevant model of polymicrobial sepsis (cecal ligation and puncture), DJ-1-/- mice had improved survival and bacterial clearance. DJ-1-/- macrophages exhibited enhanced phagocytosis and bactericidal activity in vitro, and adoptive transfer of DJ-1-/- bone marrow-derived mononuclear cells rescued wild-type mice from cecal ligation and puncture-induced mortality. In stimulated BMMs, DJ-1 inhibited ROS production by binding to p47phox, a critical component of the NADPH oxidase complex, disrupting the complex and facilitating Nox2 (gp91phox) ubiquitination and degradation. Knocking down DJ-1 (siRNA) in THP-1 (human monocytic cell line) and polymorphonuclear cells from patients with sepsis enhanced bacterial killing and respiratory burst. DJ-1 protein levels were elevated in plasma from patients with sepsis. Higher levels of circulating DJ-1 were associated with increased organ failure and death. CONCLUSIONS: These novel findings reveal DJ-1 impairs optimal ROS production for bacterial killing with important implications for host survival in sepsis.

ATF3 protects pulmonary resident cells from acute and ventilator-induced lung injury by preventing Nrf2 degradation.

AIMS: Ventilator-induced lung injury (VILI) contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). Absence of activating transcription factor 3 (ATF3) confers susceptibility to ALI/VILI. To identify cell-specific ATF3-dependent mechanisms of susceptibility to ALI/VILI, we generated ATF3 chimera by adoptive bone marrow (BM) transfer and randomized to inhaled saline or lipopolysacharide (LPS) in the presence of mechanical ventilation (MV). Adenovirus vectors to silence or overexpress ATF3 were used in primary human bronchial epithelial cells and murine BM-derived macrophages from wild-type or ATF3-deficient mice. RESULTS: Absence of ATF3 in myeloid-derived cells caused increased pulmonary cellular infiltration. In contrast, absence of ATF3 in parenchymal cells resulted in loss of alveolar-capillary membrane integrity and increased exudative edema. ATF3-deficient macrophages were unable to limit the expression of pro-inflammatory mediators. Knockdown of ATF3 in resident cells resulted in decreased junctional protein expression and increased paracellular leak. ATF3 overexpression abrogated LPS induced membrane permeability. Despite release of ATF3-dependent Nrf2 transcriptional inhibition, mice that lacked ATF3 expression in resident cells had increased Nrf2 protein degradation. INNOVATION: In our model, in the absence of ATF3 in parenchymal cells increased Nrf2 degradation is the result of increased Keap-1 expression and loss of DJ-1 (Parkinson disease [autosomal recessive, early onset] 7), previously not known to play a role in lung injury. CONCLUSION: Results suggest that ATF3 confers protection to lung injury by preventing inflammatory cell recruitment and barrier disruption in a cell-specific manner, opening novel opportunities for cell specific therapy for ALI/VILI.

Effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice.

OBJECTIVE: Recent evidence suggests that mesenchymal stem cells may attenuate lung inflammation and fibrosis in acute lung injury. However, so far, no study has investigated the effects of mesenchymal stem cell therapy on the time course of the structural, mechanical, and remodeling properties in pulmonary or extrapulmonary acute lung injury. DESIGN: Prospective randomized controlled experimental study. SETTING: University research laboratory. SUBJECTS: One hundred forty-three females and 24 male C57BL/6 mice. INTERVENTIONS: Control mice received saline solution intratracheally (0.05 mL, pulmonary control) or intraperitoneally (0.5 mL, extrapulmonary control). Acute lung injury mice received Escherichia coli lipopolysaccharide intratracheally (2 mg/kg in 0.05 mL of saline/mouse, pulmonary acute lung injury) or intraperitoneally (20 mg/kg in 0.5 mL of saline/mouse, extrapulmonary acute lung injury). Mesenchymal stem cells were intravenously injected (IV, 1 × 10 cells in 0.05 mL of saline/mouse) 1 day after lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: At days 1, 2, and 7, static lung elastance and the amount of alveolar collapse were similar in pulmonary and extrapulmonary acute lung injury groups. Inflammation was markedly increased at day 2 in both acute lung injury groups as evidenced by neutrophil infiltration and levels of cytokines in bronchoalveolar lavage fluid and lung tissue. Conversely, collagen deposition was only documented in pulmonary acute lung injury. Mesenchymal stem cell mitigated changes in elastance, alveolar collapse, and inflammation at days 2 and 7. Compared with extrapulmonary acute lung injury, mesenchymal stem cell decreased collagen deposition only in pulmonary acute lung injury. Furthermore, mesenchymal stem cell increased metalloproteinase-8 expression and decreased expression of tissue inhibitor of metalloproteinase-1 in pulmonary acute lung injury, suggesting that mesenchymal stem cells may have an effect on the remodeling process. This change may be related to a shift in macrophage phenotype from M1 (inflammatory and antimicrobial) to M2 (wound repair and inflammation resolution) phenotype. CONCLUSIONS: Mesenchymal stem cell therapy improves lung function through modulation of the inflammatory and remodeling processes. In pulmonary acute lung injury, a reduction in collagen fiber content was observed associated with a balance between metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 expressions.

Network analysis of transcriptional responses induced by mesenchymal stem cell treatment of experimental sepsis.

Although bone marrow-derived mesenchymal stem cell (MSC) systemic administration reduces sepsis-associated inflammation, organ injury, and mortality in clinically relevant models of polymicrobial sepsis, the cellular and molecular mechanisms mediating beneficial effects are controversial. This study identifies the molecular mechanisms of MSC-conferred protection in sepsis by interrogating transcriptional responses of target organs to MSC therapy. Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture, followed 6 hours later by an i.v. injection of either MSCs or saline. Total RNA from lungs, hearts, kidneys, livers, and spleens harvested 28 hours after cecal ligation and puncture was hybridized to mouse expression bead arrays. Common transcriptional responses were analyzed using a network knowledge-based approach. A total of 4751 genes were significantly changed between placebo- and MSC-treated mice (adjusted P ≤ 0.05). Transcriptional responses identified three common effects of MSC administration in all five organs examined: i) attenuation of sepsis-induced mitochondrial-related functional derangement, ii down-regulation of endotoxin/Toll-like receptor innate immune proinflammatory transcriptional responses, and iii) coordinated expression of transcriptional programs implicated in the preservation of endothelial/vascular integrity. Transcriptomic analysis indicates that the protective effect of MSC therapy in sepsis is not limited to a single mediator or pathway but involves a range of complementary activities affecting biological networks playing critical roles in the control of host cell metabolism and inflammatory response.

Salutary effect of resveratrol on sepsis-induced myocardial depression.

OBJECTIVES: We hypothesized that resveratrol administration would reverse sepsis-dependent downregulation of peroxisome proliferator activated receptor-γ coactivator 1α, preserve mitochondrial integrity, and rescue animals from sepsis-induced myocardial failure. SETTING: Teaching hospital research laboratory. INTERVENTIONS: Cecal ligation and puncture in mice was performed to induce sepsis. Mice that underwent cecal ligation and puncture were randomly assigned to receive resveratrol (30 mg/kg or 60 mg/kg) or vehicle 1 mL sodium chloride 0.9% subcutaneously in the scruff of the neck directly after surgery and at 16, 24, and 40 hrs, respectively. MEASUREMENTS AND RESULTS: Forty-eight hrs after cecal ligation and puncture, cardiac performance was established using echocardiography. Mitochondrial integrity was evaluated with electron microscopy, and changes in gene expression were evaluated with microarray analysis. Survival at 48 hrs was just under 50% and comparable between groups. Myocardial contractile function significantly improved after resveratrol treatment. Resveratrol-treated mice developed focal areas of edema, whereas vehicle-treated mice developed significant, diffuse myocardial edema. Electron microscopy revealed widespread swollen mitochondria with ruptured outer membranes, autophagosomes, and vacuolation of the internal compartment, which were significantly attenuated in resveratrol-treated animals. Resveratrol treatment significantly increased cardiac expression of peroxisome proliferator-activated receptor-γ coactivator 1a. Microarray analysis revealed that resveratrol treatment resulted in upregulation of the peroxisome proliferator-activated receptor-γ coactivator gene set containing genes known to be regulated by this transcriptional coactivator. Our data strongly suggest that administration of resveratrol modulates bioenergy metabolism, substrate utilization, oxidative stress, and detoxification pathways associated with both mitochondrial and cardiac pathological conditions, but does not alter mortality from sepsis. CONCLUSIONS: The salutary effects of resveratrol on cecal ligation and puncture-induced myocardial dysfunction are associated with increased peroxisome proliferator-activated receptor-γ coactivator 1a abundance and function. Preservation of myocardial energy production capacity, prevention of secondary injury, mitigation of inflammation, and reversal of sepsis-induced myocardial remodeling are likely to underlie its beneficial effects. This however, does not result in improved survival.

Neuroimmune regulation of ventilator-induced lung injury.

RATIONALE: Ventilator-induced lung injury (VILI) contributes to the mortality in patients with acute lung injury by increasing inflammation. Recent evidence suggests that stimulation of the cholinergic antiinflammatory pathway may be an attractive way to attenuate inflammatory injury. OBJECTIVES: To determine the role of vagus nerve signaling in VILI and establish whether stimulation of the vagus reflex can mitigate VILI. METHODS: We performed bilateral vagotomy in a mouse model of high-tidal volume-induced lung injury. We performed pharmacological and electrical vagus nerve stimulation in a rat model of VILI following ischemia/reperfusion injury. To determine the contribution of the alpha 7 acetylcholine nicotinic receptor to pulmonary cell injury, we exposed human bronchial epithelial cells to cyclic stretch in the presence of specific agonist or antagonist of the alpha 7 receptor. MEASUREMENTS AND MAIN RESULTS: Vagotomy exacerbates lung injury from VILI in mice as demonstrated by increased wet-to-dry ratio, infiltration of neutrophils, and increased IL-6. Vagal stimulation attenuates lung injury in rats after ischemia/reperfusion injury ventilated with high-volume strategies. Treatment of both mice and rats with the vagus mimetic drug semapimod resulted in decreased lung injury. Vagotomy also increased pulmonary apoptosis, whereas vagus stimulation (electrical and pharmacological) attenuated VILI-induced apoptosis. In vitro studies suggest that vagus-dependent effects on inflammation and apoptosis are mediated via the α7 nicotinc acetylcholine receptor-dependent effects on cyclic stretch-dependent signaling pathways c-jun N-terminal kinase and tumor necrosis factor receptor superfamily, member 6. CONCLUSIONS: Stimulation of the cholinergic antiinflammatory reflex may represent a promising alternative for the treatment of VILI.

Differential regulation of NMDA receptor function by DJ-1 and PINK1.

Dysfunction of PTEN-induced kinase 1 (PINK1) or DJ-1 promotes neuronal death and is implicated in the pathogenesis of Parkinson's disease, but the underlying mechanisms remain unclear. Given the roles of N-methyl-d-aspartate receptor (NMDAr)-mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ-1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr-mediated whole-cell currents by enhancing the function of NR2A-containing NMDAr subtype (NR2ACNR). However, DJ-1 decreased NMDAr-mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B-containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ-1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ-1-dependent signal pathways DJ-1/PTEN/NR2BCNR and DJ-1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ-1, while promoted NMDA-induced neuronal death through the overactivation of PTEN/NR2BCNR-dependent cell death pathway, induced a neuroprotective effect to counteract DJ-1 dysfunction-mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival-promoting pathway. Thus, PINK1 acts with DJ-1 in a common pathway to regulate NMDAr-mediated neuronal death. This study suggests that the DJ-1/PTEN/NR2BCNR and DJ-1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson's disease.

Activating transcription factor 3 confers protection against ventilator-induced lung injury.

RATIONALE: Ventilator-induced lung injury (VILI) significantly contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury. Understanding the molecular basis for response to cyclic stretch (CS) and its derangement during high-volume ventilation is of high priority. OBJECTIVES: To identify specific molecular regulators involved in the development of VILI. METHODS: We undertook a comparative examination of cis-regulatory sequences involved in the coordinated expression of CS-responsive genes using microarray analysis. Analysis of stretched versus nonstretched cells identified significant enrichment for genes containing putative binding sites for the transcription factor activating transcription factor 3 (ATF3). To determine the role of ATF3 in vivo, we compared the response of ATF3 gene-deficient mice to wild-type mice in an in vivo model of VILI. MEASUREMENTS AND MAIN RESULTS: ATF3 protein expression and nuclear translocation is increased in the lung after mechanical ventilation in wild-type mice. ATF3-deficient mice have greater sensitivity to mechanical ventilation alone or in conjunction with inhaled endotoxin, as demonstrated by increased cell infiltration and proinflammatory cytokines in the lung and bronchoalveolar lavage, and increased pulmonary edema and indices of tissue injury. The expression of stretch-responsive genes containing putative ATF3 cis-regulatory regions was significantly altered in ATF3-deficient mice. CONCLUSIONS: ATF3 deficiency confers increased sensitivity to mechanical ventilation alone or in combination with inhaled endotoxin. We propose ATF3 acts to counterbalance CS and high volume-induced inflammation, dampening its ability to cause injury and consequently protecting animals from injurious CS.

Preservation of GABAA receptor function by PTEN inhibition protects against neuronal death in ischemic stroke.

BACKGROUND AND PURPOSE: Downregulation of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is thought to be a novel neuroprotective strategy in ischemic stroke, but the underlying mechanisms remain unclear. In this study, we aimed to validate the use of PTEN regulation of gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) as a molecular target for the treatment of ischemic stroke. Because suppression of GABA(A)Rs contributes to ischemic neuron death, describing the intracellular signaling that interacts with GABA(A)Rs in ischemic neurons would provide a molecular basis for novel stroke therapies. METHODS: We measured surface GABA(A)R expression by immunocytochemical labeling and surface protein biotinylation assay. Knockdown and overexpression approaches were used to test the effects of PTEN on the expression and function of GABA(A)Rs. Neuronal death was detected in both in vitro and in vivo stroke models. RESULTS: The knockdown and overexpression approaches provided the first evidence that PTEN negatively regulated membrane expression and function of GABA(A)Rs in rat hippocampal neurons. Importantly, we demonstrated that a PTEN inhibitor prevented the reduction of surface GABA(A)Rs in injured hippocampal neurons subjected to oxygen-glucose deprivation, an in vitro insult that mimics ischemic injury, whereas a GABA(A)R antagonist significantly reduced this PTEN inhibitor-induced neuroprotection in both the in vitro and in vivo ischemic stroke models. CONCLUSIONS: Our study provides direct evidence that downregulation of PTEN protects against ischemic neuron death by preserving GABA(A)R function. Targeting this pathway may be an effective strategy for development of selective, potent stroke treatments.

Regulation of PINK1 by NR2B-containing NMDA receptors in ischemic neuronal injury.

Dysfunction of PTEN-induced kinase-1 (PINK1) is implicated in neurodegeneration. We report here that oxygen-glucose deprivation (OGD), an in vitro insult mimicking ischemic neuron injury, resulted in a significant reduction of PINK1 protein expression in cultured cortical neurons. The decrease of PINK1 expression was blocked by the antagonists of NMDA receptors. We revealed that the overactivation of NR2B-containing NMDA receptors (NR2BRs) was responsible for the OGD-induced PINK1 reduction. The overactivated NR2BRs also inhibited the phosphorylation, but not the protein expression, of the cell survival-promoting kinase Akt after OGD insult, indicating that OGD-induced reduction of PINK1 protein is specific in the injury paradigm. We further showed that enhancing the protein expression of PINK1 antagonized OGD-induced reduction of Akt phosphorylation, suggesting that Akt may be a downstream target of PINK1 in ischemic neuron injury. Importantly, we provided evidence that both NR2BR antagonist and PINK1 over-expression protected against OGD-induced neuronal death. These results suggest that the overactivation of NR2BRs may contribute to ischemic neuron death through suppressing PINK1-dependent survival signaling. Thus, selectively antagonizing NR2BR signal pathway-induced neurotoxicity may be a potential neuroprotection strategy.

Oleylethanolamide activates Ras-Erk pathway and improves myocardial function in doxorubicin-induced heart failure.

Oleylethanolamide (OEA) is a natural fatty acid ethanolamide produced in the heart, but its biological actions in myocardium have not yet been defined. This study was carried out to determine whether OEA could be used to prevent the development of heart failure or improve evolving heart failure. We studied in vivo and in vitro actions of OEA in cardiac muscle. In an animal model of doxorubicin cardiomyopathy, OEA showed robust effects and attenuated the progression of systolic/diastolic dysfunction and ventricular remodeling. During evolving doxorubicin cardiomyopathy, a therapeutic course of OEA treatment partially restored myocardial function. The preventive and therapeutic effects of OEA were associated with significant improvement of survival. To investigate the mechanism of OEA action in cardiac muscle, we have carried out in vitro experiments in cultured cardiomyocytes. The results showed that OEA, through activation of Ras-Raf-1-Mek-Erk signaling, inhibited doxorubicin-induced apoptosis. Additional experiments showed that OEA activation of the Erk pathway involved activation of Neu/ErbB2 receptor, which suggests OEA actions in cardiac muscle might require activation of Neu/ErbB2. In summary, OEA improved ventricular remodeling and augmented cardiac function in doxorubicin cardiomyopathy, possibly involving activation of Neu/ErbB2 and Ras-Erk signaling. These findings suggest OEA is a novel cardioprotective compound that may be used to develop new strategies for the management of cardiomyopathy.

Insulin deficiency downregulated heat shock protein 60 and IGF-1 receptor signaling in diabetic myocardium.

Heat shock protein (Hsp)60 and IGF-1 receptor signaling protect cardiac muscle against injury. The abundance of cardiac IGF-1 receptor can be upregulated by Hsp60, but how diabetes modulates cardiac muscle Hsp60 has not yet been defined. We investigated the changes of Hsp60 and IGF-1 receptor signaling in the diabetic myocardium and studied how diabetes modulates Hsp60 and IGF-1 receptor in diabetic myocardium. In the streptozotocin (STZ)-induced diabetic rat, downregulation of Hsp60 and IGF-1 receptor occurred 4 days after induction of diabetes. IGF-1 activation of IGF-1 receptor, Mek, and Akt were reduced accordingly in the diabetic myocardium. The independent effect of insulin and hyperglycemia on Hsp60 was investigated in primary cardiomyocytes. Incubating cardiomyocytes with insulin was associated with dose-dependent increase of Hsp60 protein. In contrast, the abundance of Hsp60 was not affected by high concentration of glucose in these cells. To further determine the independent effects of hyperglycemia and insulin deficiency on the changes of myocardial Hsp60 and IGF-1 receptor, we used phlorizin to normalize blood glucose in diabetic rats. In the phlorizin-treated diabetic rats, myocardial Hsp60 was lower than that of the normal controls. In contrast, insulin treatment normalized myocardial Hsp60 in the diabetic rats. Because phlorizin does not alter insulin secretion, Hsp60 expression was modulated by insulin and not by hyperglycemia. Similar changes of Hsp60 and IGF-1 receptor were observed in the skeletal muscle of STZ-induced diabetic rats. These findings suggest that insulin deficiency is a novel mechanism that leads to downregulation of Hsp60 in diabetic muscle tissues. The development of diabetic cardiomyopathy might have involved downregulation of Hsp60 and subsequent reduction of IGF-1 receptor signaling.

Using DNA microarray to identify Sp1 as a transcriptional regulatory element of insulin-like growth factor 1 in cardiac muscle cells.

High throughput gene expression profiling with DNA microarray provides an opportunity to analyze transcriptional regulation of hundreds or thousands of similarly regulated genes. Transcriptional regulation of gene expression plays an important role in myocardial remodeling. We have studied cardiac muscle gene expression with DNA microarray and used a computational strategy to identify common promoter motifs that respond to insulin-like growth factor 1 (IGF-1) stimulation in cardiac muscle cells. The analysis showed that the Sp1 binding site is a likely target of IGF-1 action. Further experiments with gel shift assay indicated that IGF-1 regulated the Sp1 site in cardiomyocytes, by increasing the abundance of Sp1 and Sp3 proteins. Using firefly luciferase as reporter gene, additional experiments showed that IGF-1 activated the promoter of cyclin D3 and Glut1. Both promoters contain one Sp1 site. The effect of IGF-1 on these two promoters was abolished with siRNA for Sp1. Thus, the transcriptional activation of these two promoters by IGF-1 requires the induction of Sp1 protein. These experiments suggest that the global transcriptional regulatory actions of IGF-1 involve activation of the Sp1 site in cardiac muscle. The computational model we have developed is a prototypical method that may be further developed to identify unique cis- and trans-acting elements in response to hormonal stimulation during cardiac muscle growth, repair, and remodeling in normal and abnormal cardiac muscle.

Hsp10 and Hsp60 suppress ubiquitination of insulin-like growth factor-1 receptor and augment insulin-like growth factor-1 receptor signaling in cardiac muscle: implications on decreased myocardial protection in diabetic cardiomyopathy.

We have investigated the effects of two heat shock proteins, Hsp10 and Hsp60, on insulin-like growth factor-1 receptor (IGF-1R) signaling in cardiac muscle cells. Neonatal cardiomyocytes were transduced with Hsp10 or Hsp60 via adenoviral vector. Compared with the cells transduced with a control vector, overexpression of Hsp10 or Hsp60 increased the abundance of IGF-1R and IGF-1-stimulated receptor autophosphorylation. Thus, Hsp10 and Hsp60 overexpression increased the number of functioning receptors and amplified activation of IGF-1R signaling. IGF-1 stimulation of MEK, Erk, p90Rsk, and Akt were accordingly augmented. Transducing cardiomyocytes with antisense Hsp60 oligonucleotides reduced Hsp60 expression, decreased the abundance of IGF-1R, attenuated IGF-1R autophosphorylation, and suppressed the pro-survival action of IGF-1 in cardiomyocytes. Using cycloheximide to inhibit protein synthesis did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60. Additional experiments showed that Hsp10 and Hsp60 suppressed polyubiquitination of IGF-1 receptor. These data indicate that Hsp10 and Hsp60 can modulate IGF-1R signaling through post-translational modification. In animal models of diabetes, diabetic myocardium is associated with decreased abundance of Hsp60, increased ubiquitination of IGF-1R, and lower level of IGF-1R protein. Declined myocardial protection is a major feature of diabetic cardiomyopathy. These data suggest that decreased Hsp60 expression and subsequent decline of IGF-1R signaling may be a fundamental mechanism underlying the development of diabetic cardiomyopathy.