Nucelosides such as adenosine (Ado) influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides liberated at local sites of inflammation are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases including ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73), found on the surface of a variety of cell types. Once generated, Ado is made available to bind and activate one of four G-protein-coupled Ado receptors. Recent in vitro and in vivo studies implicate Ado in a broad array of tissue protective mechanisms that provide new insight into adenosine actions. Studies in cultured cells and murine tissues have indicated that Ado receptors couple to novel post-translational protein modifications, including Cullin deneddylation, as a new anti-inflammatory mechanism. Studies in Ado receptor-null mice have been revealing and indicate a particularly important role for the Ado A2B receptor in animal models of intestinal inflammation. Here, we review contributions of Ado to cell and tissue stress responses, with a particular emphasis on the gastrointestinal mucosa.
mucosa; inflammation; colitis; neutrophil; epithelium; endothelium; murine model
Inflammatory lesions, ischemic tissues or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors – particularly of hypoxia-inducible factor 1α (HIF1A) - results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors - the ADORA1, ADORA2A, ADORA2B and ADORA3 receptor. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides such as ATP or AMP. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors – particularly of the ADORA2B – results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia-tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.
adenosine; A1; A2A; A2B; A3; ischemia; cancer; hypoxia-inducible factor; HIF1; HIF2; equilibrative nucleoside transporters; ENT1; ENT2; adenosine kinase; adenosine deaminase; CD73; ecto-nucleotidase; CD39; apyrase; AMP; ATP; acute lung injury; colitis; inflammatory bowel disease; ischemia; sepsis
adenosine; A1; A2A; A2B; A3; ischemia; cancer; hypoxia-inducible factor; equilibrative nucleoside transporters; ENT1; ENT2; adenosine kinase; adenosine deaminase; CD73; ecto-nucleotidase; CD39; apyrase; AMP; ATP; acute lung injury; colitis; inflammatory bowel disease; ischemia; sepsis
The recent approval by the FDA of cancer vaccines and drugs that blockade immunological negative regulators has further enhanced interest in promising approaches of the immunotherapy of cancer. However, the disappointingly short life extension has also underscored the need to better understand the mechanisms that prevent tumor rejection and survival even after the blockade of immunological negative regulators. Here, we describe the implications of the “metabolism-based” immunosuppressive mechanism, where the local tissue hypoxia-driven accumulation of extracellular adenosine triggers suppression via A2 adenosine receptors on the surface of activated immune cells. This molecular pathway is of critical importance in mechanisms of immunosuppression in inflamed and cancerous tissue microenvironments. The protection of tumors by tumor-generated extracellular adenosine and A2 adenosine receptors could be the misguided application of the normal tissue-protecting mechanism that limits excessive collateral damage to vital organs during the anti-pathogen immune response. The overview of the current state of the art regarding the immunosuppressive effects of extracellular adenosine is followed by an historical perspective of studies focused on the elucidation of the physiological negative regulators that protect tissues of vital organs from excessive collateral damage, but, as a trade-off, may also weaken the anti-pathogen effector functions and negate the attempts of anti-tumor immune cells to destroy cancerous cells.
adenosine; A2A adenosine receptor; cyclic AMP; hypoxia; inflammation; tumor; cancer immunotherapy; adoptive immunotherapy; tumor microenvironment; immunosuppression; T lymphocytes; regulatory T cells; cytokines; cytotoxicity
Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis.
adenosine receptors; inflammation; fibrosis; vascular barrier function; CD73; ADORA2B; ADORA2A; ADORA3; ADORA1; acute lung injury; remodeling; anti-inflammatory
Though the existence of H2S in biological tissues has been known for over 300 years, it is the most recently appreciated of the gasotransmitters as a physiologic messenger molecule. The enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) had long been speculated to generate H2S, and inhibitors of these enzymes had been employed to characterize influences of H2S in various organs. Definitive evidence that H2S is a physiologic regulator came with the development of mice with targeted deletion of CSE and CBS. Best characterized is the role of H2S, formed by CSE, as an endothelial derived relaxing factor that normally regulates blood pressure by acting through ATP-sensitive potassium channels. H2S participates in various phases of the inflammatory process, predominantly exerting anti-inflammatory actions. Currently, the most advanced efforts to develop therapeutic agents involve the combination of H2S donors with non-steroidal anti-inflammatory drugs (NSAIDs). The H2S moiety provides cytoprotection to gastric mucosa normally adversely affected by NSAIDs, while the combination of H2S and inhibition of prostaglandin synthesis may afford synergistic anti-inflammatory influences.
sulfhydration; cardio protection; inflammation; cystathionine γ-lyase; cystathionine-β-synthase
In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel-Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzymes genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wildtype VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc but not Pdk2 was decreased and skeletal muscle expression of Glut1, Pdk1 and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.
VHL; hypoxia inducible factors; glucose; insulin; glycolysis; gluconeogenesis
Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptor death receptor 4 (DR4) have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation and leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MRs) clustering and formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, TRAIL triggered ASM translocation, ceramide production and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking and fusion with membrane and formation of MR redox signaling platforms, which may play an important role in DR4-mediated redox signaling in CAECs and consequent endothelial dysfunction.
TRAIL; lysosome fusion; acid sphingomyelinase; membrane raft; endothelial cell; vasorelaxation
A major hurdle for hematopoietic stem cell (HSC) gene therapy for inherited bone marrow disorders, including Fanconi anemia (FA), is adequate engraftment of gene-modified cells. A phenotypic defect in DNA repair renders FA patients sensitive to alkylating agents such as cyclophosphamide (Cy); however, at lower doses Cy is well tolerated in the FA transplant setting. We tested whether non-alkylating agents could replace Cy for pre-transplant conditioning to enhance engraftment of FANCA gene-modified hematopoietic cells. We compared Cy pre-conditioning with fludarabine (Flu) or cytarabine (AraC) or no conditioning as a control in fanca−/− mutant mice receiving gene-modified bone marrow cells. Only mice conditioned with Cy exhibited appreciable engraftment of gene-modified cells by PCR and resistance to mitomycin C (MMC). Cy administration following transplantation increased gene marking levels in all animals treated, but highest gene marking and corresponding MMC resistance were observed in mice receiving Cy pre- and post-transplantation. Importantly, no cytogenetic abnormalities were observed in Cy-treated mice. We conclude that Cy is an effective and superior preparative regimen with respect to engraftment of lentivirus-transduced cells and functional correction in fanca−/− mice. Thus, appropriately dosed Cy may provide a suitable conditioning regimen for FA patients undergoing HSC gene therapy.
gene therapy; Fanconi anemia; lentivirus vector; cyclophosphamide; autologous transplantation
Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-β1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-β signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-β1-induced renal fibrosis. In the obstructed kidneys and TGF-β1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-β signaling by inhibiting N-linked glycosylation of the type II TGF-β receptor (TβRII), leading to an inefficient trafficking of TβRII to the membrane surface. Defective N-glycosylation of TβRII further suppressed the TGF-β1-binding to TβRII, thereby decreasing TGF-β signaling. Notably, GS-HCl treatment significantly reduced TGF-β1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-β signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-β1-induced renal fibrosis in kidney diseases.
Glucosamine-mediated attenuation of TGF-β signaling ameliorates renal fibrosis in vivoTGF-β1-induced fibrogenic action is reduced by glucosamine in vitroN-glycosylation of the type II TGF-β receptor is suppressed by glucosamineGlucosamine-induced defective N-glycosylation of TβRII decreases TGF-β signaling.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1086-1) contains supplementary material, which is available to authorized users.
Renal fibrosis; Glucosamine hydrochloride; TGF-β signaling; N-glycosylation; Type II TGF-β receptor
Human CD4+CD25highFOXP3+ T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4+CD25neg RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4+CD25highFOXP3+ Treg±150 or ±1,000 IU/ mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p<0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4+CD25high Treg and CD4+CD25neg RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme–perforin pathway depending on IL-2 concentrations.
Human CD4+CD25high Treg; Cell death; Granzymes; Perforin; Interleukin-2
Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin-receptor (IR) signaling and, when over-expressed, induces insulin resistance in vitro and in vivo. Understanding the regulation of ENPP1 expression may, thus, unravel new molecular mechanisms of insulin resistance. Recent data point to a pivotal role of the ENPP1 3’UTR, in modulating ENPP1 mRNA stability and expression. We sought to identify trans-acting proteins binding the ENPP1-3’UTR and to investigate their role on ENPP1 expression and on IR signaling. By RNA electrophoresis mobility shift analysis and tandem mass spectrometry, we demonstrated the binding of heat shock protein 70 (HSP70) to ENPP1-3’UTR. Through this binding, HSP70 stabilizes ENPP1 mRNA and increases ENPP1 transcript and protein levels. This positive modulation of ENPP1 expression is paralleled by a reduced insulin-induced IR and IRS-1 phosphorylation. Taken together these data suggest that HSP70, by affecting ENPP1 expression, may be a novel mediator of altered insulin signaling.
Inhibitors of insulin signaling; Insulin resistance; 3′untranslatedregions; Tyrosine-kinasereceptors
To test our hypothesis that transgenic induction of miR-210 in mesenchymal stem cells (MSC) simulate the pro-survival effects of ischemic preconditioning (IPC) and that engraftment of PCMSC help in functional recovery of ischemic heart by miR-210 transfer to host cardiomyocytes through gap junctions.
Methods and Results
miR-210 expression in MSC was achieved by IPC or nano-particle based transfection of miR-210 plasmid (miRMSC), and functional recovery of the infarcted heart of rat transplanted with PCMSC or miRMSC were evaluated. Both PCMSC and miRMSC showed higher survival under lethal anoxia as compared to non-PCMSC and scramble-transfected MSC (ScMSC) controls with concomitantly lower CASP8AP2 expression. Similarly, both PCMSC and miRMSC survived better and accelerated functional recovery of ischemic heart post-transplantation. To validate our hypothesis that MSC deliver miR-210 to host cardiomyocytes, in vitro co-culture between cardiomyocytes and PCMSC or miRMSC (using non-PCMSC or ScMSC as controls) showed co-localization of miR-210 with gap-junctional connexin-43. MiR-210 transfer to cardiomyocytes was blocked by heptanol pre-treatment. Moreover, higher survival of cardiomyocytes co-cultured with PCMSC was observed with concomitant expression of CASP8AP2 as compared to cardiomyocytes co-cultured with non-PCMSC thus suggesting that miR-210 was translocated from MSC to protect host cardiomyocytes.
Induction of miR-210 in MSC promoted their survival post-engraftment in the infarcted heart. Moreover, direct transfer of pro-survival miR-210 from miRMSC to host cardiomyocytes led to functional recovery of the ischemic heart.
Connexin-43; miR-210; myocardium; preconditioning; stem cells
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.
Hypoxia inducible factor-1 (HIF-1); innate immunity; Staphylococcus aureus; bacterial infection; antibiotic-resistant bacteria
Thrombin is the protease involved in blood coagulation. Its deregulation can lead to hemostatic abnormalities, which range from subtle subclinical to serious life-threatening coagulopathies, i.e., during septicemia. Additionally, thrombin plays important roles in many (patho)physiological conditions that reach far beyond its well-established role in stemming blood loss and thrombosis, including embryonic development and angiogenesis but also extending to inflammatory processes, complement activation, and even tumor biology. In this review, we will address thrombin’s broad roles in diverse (patho)physiological processes in an integrative way. We will also discuss thrombin as an emerging major target for novel therapies.
Coagulation; Inflammation; Hemostasis; Cancer; Thrombin (F2); Thrombosis; Immune system; Metastasis; Angiogenesis; Invasion; Prognosis; Trousseau’s syndrome
Cardiac hypertrophy is a response of the myocardium to increased workload and is characterised by an increase of myocardial mass and an accumulation of extracellular matrix (ECM). As an ECM protein, an integrin ligand, and an angiogenesis inhibitor, all of which are key players in cardiac hypertrophy, mindin is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure. In this study, we investigated the role of mindin in cardiac hypertrophy using littermate Mindin knockout (Mindin−/−) and wild-type (WT) mice. Cardiac hypertrophy was induced by aortic banding (AB) or angiotensin II (Ang II) infusion in Mindin−/− and WT mice. The extent of cardiac hypertrophy was quantitated by echocardiography and by pathological and molecular analyses of heart samples. Mindin−/− mice were more susceptible to cardiac hypertrophy and fibrosis in response to AB or Ang II stimulation than wild type. Cardiac function was also markedly exacerbated during both systole and diastole in Mindin−/− mice in response to hypertrophic stimuli. Western blot assays further showed that the activation of AKT/glycogen synthase kinase 3β (GSK3β) signalling in response to hypertrophic stimuli was significantly increased in Mindin−/− mice. Moreover, blocking AKT/GSK3β signalling with a pharmacological AKT inhibitor reversed cardiac abnormalities in Mindin−/− mice. Our data show that mindin, as an intrinsic cardioprotective factor, prevents maladaptive remodelling and the transition to heart failure by blocking AKT/GSK3β signalling.
Mindin; Hypertrophy; Remodelling; Signal transduction; AKT
Myocardial infarction is a major cause of morbidity and mortality in the developing and developed world. Although current interventions have been successful in prolonging life, they are inadequate because mortality is still high among MI patients. The multifunctional Ca2+/calmodulin dependent protein kinase (CaMKII) plays a key role in the structure and contractility of the myocardium. CaMKII activity is increased in MI hearts and CaMKII promotes cardiac hypertrophy and inflammation, processes consistently activated by myocardial injury. Hypertrophy and inflammation are also related to neurohumoral and redox signaling that uncouple CaMKII activation from Ca2+/calmodulin dependence. Thus, CaMKII may act as a nodal point for integrating hypertrophic and inflammatory signaling in myocardium.
CaMKII; Heart; Myocardium; Myocardial infarction; Heart attack; Inflammation; Hypertrophy; Toll-like receptors; Oxidative stress; ROS; AngII
Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling—for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.
Reactive oxygen species (ROS); Mitochondria; ROS signaling; ROS indicators; In vivo imaging
Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1*) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1* expressed in live GAS and tested whether the modulation of M1–fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1* showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1–fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1–fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1–fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1.
Group A Streptococcus; M protein; Fibrinogen; Host cell interactions; Bacterial virulence
Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of the hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through induction of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the expression of LOX and LOXL proteins, collagen cross-linking, CD11b+ BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1α-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor.
Hypoxia-inducible factor 1; Lysyl oxidase; Bone marrow derived cells; Breast cancer metastasis; Metastatic niche; HIF inhibitor
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
Microvascular ischemia and infections are associated with the development of chronic rejection following lung transplantation. The von Hippel–Lindau protein (VHL) controls protein levels of hypoxia-inducible factors (HIFs), regulates vascular repair, and improves tissue perfusion. Here, we studied the role of VHL in microvascular repair by orthotopically transplanting tracheas into mice with VHL haplodeficiency in Tie2 lineage cells. We showed that VHL haplodeficiency prolonged airway microvascular perfusion and promoted tissue blood flow through the production of the angiogenic factors, SDF-1 and angiopoietin 1. VHL-haplodeficient pulmonary endothelial cells exhibited increased angiogenic activity, resistance to serum deprivation-induced cell death, and enhanced microvascular repair. By contrast, in recipient mice with HIF-1α deficiency in Tie2 lineage cells, microvascular repair was significantly diminished and suggested that recipient-derived HIF-1α normally participates in the repair of alloimmune-mediated microvascular damage. To evaluate the translational impact of our findings, we compared VHL-haplodeficient mice with wild-type controls using a model of Aspergillus airway infection. In 83 % of the VHL-haplodeficient recipients, Aspergillus fumigatus was noninvasive in contrast to 75 % of wild-type mice in which the mold was deeply invasive. Our study demonstrated that stabilization of HIF-1α in angiogenic cells, through Tie2 cell VHL haplodeficiency, promoted airway microvascular regeneration and vascular normalization and thereby minimized tissue ischemia and hypoxia. By also mitigating the virulence of A. fumigatus, a common pathogen and itself a risk factor for the development of lung transplant rejection, the selective enhancement of HIF-1α expression has the prospect of offering several novel therapeutic effects to transplant recipients.
Microvascular loss and prolonged ischemia occurs with acute rejection.Von Hippel-Lindau (VHL) protein controls hypoxia inducible factors (HIFs).In tracheal allografts, VHL haplodeficient Tie2 cells promote neovascularization.Reduced transplant ischemia limits Aspergillus invasion.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1063-8) contains supplementary material, which is available to authorized users.
HIF-1α; Aspergillus fumigatus; VHL; Orthotopic tracheal transplant; Obliterative bronchiolitis; Chronic lung transplant rejection; Microvascular perfusion
Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1006-4) contains supplementary material, which is available to authorized users.
Rab escort protein 1; Gene therapy; Choroideremia; Rab GTPase; Retinitis pigmentosa; AAV
Endothelial progenitor cells (EPCs); Pulmonary arterial hypertension (PAH); High-throughput testing; Surface markers; Microfluidic device
Thalidomide is experimentally used to treat various human cancers; however, clinical responses to thalidomide are sporadic. Here we demonstrate that CUL4A plays an oncogenic role in prostate cancer development and prostate cancer cells with higher level of CUL4A are particularly sensitive to thalidomide treatment. We show that CUL4A is frequently overexpressed in human primary prostate cancer and cell lines. Notably, subjects with tumors that highly expressed CUL4A had poor overall survival. CUL4A downregulation inhibited cell proliferation and induced apoptosis in vitro and in vivo, whereas CUL4A overexpression transformed human normal prostate epithelial cells and promoted invasion, which was attenuated by the extracellular signal-regulated kinase (ERK) inhibitor. We further show that the sensitivity to thalidomide is positively correlated with CUL4A expression in a panel of prostate cell lines. Ectopic CUL4A expression greatly enhanced sensitivity to thalidomide, while its downregulation conferred resistance to this drug. Mechanistically, thalidomide decreased CUL4A in a time- and dose-dependent manner, consequently leading to inaction of ERK pathway. Finally, we show that cereblon level is correlated with CUL4A expression and downregulated in thalidomide-resistant prostate cancer cell. Our results offer the first evidence that CUL4A is a potential therapeutic target for prostate cancer and may serve as a biomarker for assessing prognosis of human prostate cancer and response to thalidomide treatment.
CUL4A; Thalidomide; Prostate cancer; Translational research; Cereblon