Adiponectin suppresses stiffness-dependent, pro-fibrotic activation of lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible respiratory disease with limited therapeutic options. A hallmark of IPF is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We performed an unbiased, next generation sequencing (NGS) screen to identify signaling pathways involved in stiffness-dependent lung fibroblast activation. Adipocytokine signaling was downregulated in primary lung fibroblasts (PFs) cultured on stiff matrices. Re-activating adipocytokine signaling with adiponectin suppressed stiffness-dependent activation of human PFs. Adiponectin signaling depended on CDH13 expression and p38 mitogen-activated protein kinase gamma (p38MAPKγ) activation. CDH13 expression and p38MAPKγ activation were strongly reduced in lungs from IPF donors. Our data suggest that adiponectin-signaling via CDH13 and p38MAPKγ activation suppresses pro-fibrotic activation of fibroblasts in the lung. Targeting of the adiponectin signaling cascade may provide therapeutic benefits in IPF.

Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury.

Animal models are important to mimic certain pathways or biological aspects of human pathologies including acute and chronic pulmonary diseases. We developed a novel and flexible mouse model of acute epithelial lung injury based on adeno-associated virus (AAV) variant 6.2-mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial and alveolar epithelial cells can be observed. In contrast to other lung injury models, the here described mouse model provides the possibility of targeted injury using specific tropisms of AAV vectors or cell-type-specific promotors to drive the human DTR expression. Also, generation of cell-specific mouse lines is not required. Detailed characterization of the AAV-DTR/DT mouse model including titration of viral genome (vg) load and administered DT amount revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and unspecified cells) and elevation of degenerated cells and infiltrated leukocytes in lung tissue, dependent of vg load and DT dose. Cytokine levels in BAL fluid showed different patterns with higher vg load, e.g., IFNγ, TNFα, and IP10 increasing and IL-5 and IL-6 decreasing, whereas lung function was not affected. In addition, laser-capture microdissection (LCM)-based proteomics of bronchial epithelium and alveolar tissue revealed upregulated immune and inflammatory responses in all regions and extracellular matrix deposition in infiltrated alveoli. Overall, our novel AAV-DTR/DT model allows investigation of repair mechanisms following epithelial injury and resembles specific mechanistic aspects of acute and chronic pulmonary diseases.

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface model.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial-like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)-derived air-liquid interface (ALI) model of alveolar epithelial type II (ATII)-like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF-relevant cocktail (IPF-RC) to mimic the pro-fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF-RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA-seq) of these cultures reveals significant overlap with human IPF patient data. IPF-RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial-like expression signature, providing evidence that a pro-fibrotic cytokine environment can influence the proximo-distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF-associated bronchiolization of the lung epithelium in vitro.

Deep Learning-Based Detection of Endothelial Tip Cells in the Oxygen-Induced Retinopathy Model.

Proliferative retinopathies, such as diabetic retinopathy and retinopathy of prematurity, are leading causes of vision impairment. A common feature is a loss of retinal capillary vessels resulting in hypoxia and neuronal damage. The oxygen-induced retinopathy model is widely used to study revascularization of an ischemic area in the mouse retina. The presence of endothelial tip cells indicates vascular recovery; however, their quantification relies on manual counting in microscopy images of retinal flat mount preparations. Recent advances in deep neural networks (DNNs) allow the automation of such tasks. We demonstrate a workflow for detection of tip cells in retinal images using the DNN-based Single Shot Detector (SSD). The SSD was designed for detection of objects in natural images. We adapt the SSD architecture and training procedure to the tip cell detection task and retrain the DNN using labeled tip cells in images of fluorescently stained retina flat mounts. Transferring knowledge from the pretrained DNN and extensive data augmentation reduced the amount of required labeled data. Our system shows a performance comparable to the human level, while providing highly consistent results. Therefore, such a system can automate counting of tip cells, a readout frequently used in retinopathy research, thereby reducing routine work for biomedical experts.

Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial-temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4ß7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial-temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

Disease Progression and Pharmacological Intervention in a Nutrient-Deficient Rat Model of Nonalcoholic Steatohepatitis.

BACKGROUND: There is a marked need for improved animal models of nonalcoholic steatohepatitis (NASH) to facilitate the development of more efficacious drug therapies for the disease. METHODS: Here, we investigated the development of fibrotic NASH in male Wistar rats fed a choline-deficient L-amino acid-defined (CDAA) diet with or without cholesterol supplementation for subsequent assessment of drug treatment efficacy in NASH biopsy-confirmed rats. The metabolic profile and liver histopathology were evaluated after 4, 8, and 12 weeks of dieting. Subsequently, rats with biopsy-confirmed NASH were selected for pharmacological intervention with vehicle, elafibranor (30 mg/kg/day) or obeticholic acid (OCA, 30 mg/kg/day) for 5 weeks. RESULTS: The CDAA diet led to marked hepatomegaly and fibrosis already after 4 weeks of feeding, with further progression of collagen deposition and fibrogenesis-associated gene expression during the 12-week feeding period. Cholesterol supplementation enhanced the stimulatory effect of CDAA on gene transcripts associated with fibrogenesis without significantly increasing collagen deposition. Pharmacological intervention with elafibranor, but not OCA, significantly reduced steatohepatitis scores, and fibrosis-associated gene expression, however, was unable to prevent progression in fibrosis scores. CONCLUSION: CDAA-fed rats develop early-onset progressive NASH, which offers the opportunity to probe anti-NASH compounds with potential disease-modifying properties.

Animal models for the study of inflammatory bowel diseases: a meta-analysis on modalities for imaging inflammatory lesions.

Inflammatory bowel diseases are lifelong disorders affecting the gastrointestinal tract characterized by intermittent disease flares and periods of remission with a progressive and destructive nature. Unfortunately, the exact etiology is still not completely known, therefore a causal therapy to cure the disease is not yet available. Current treatment options mainly encompass the use of non-specific anti-inflammatory agents and immunosuppressive drugs that cause significant side effects that often have a negative impact on patients' quality of life. As the majority of patients need a long-term follow-up it would be ideal to rely on a non-invasive technique with good compliance. Currently, the gold standard diagnostic tools for managing IBD are represented by invasive procedures such as colonoscopy and histopathology. Nevertheless, recent advances in imaging technology continue to improve the ability of imaging techniques to non-invasively monitor disease activity and treatment response in preclinical models of IBD. Novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. Furthermore, molecular imaging advances allow us to increase our knowledge on the critical biological pathways involved in disease progression by characterizing in vivo processes at a cellular and molecular level and enabling significant improvements in the understanding of the etiology of IBD. This review presents a critical and updated overview on the imaging advances in animal models of IBD. Our aim is to highlight the potential beneficial impact and the range of applications that imaging techniques could offer for the improvement of the clinical monitoring and management of IBD patients: diagnosis, staging, determination of therapeutic targets, monitoring therapy and evaluation of the prognosis, personalized therapeutic approaches.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Pivotal role of phospholipase D1 in tumor necrosis factor-α-mediated inflammation and scar formation after myocardial ischemia and reperfusion in mice.

Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α-mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-ß secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1(-/-) mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1(-/-) mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α-mediated inflammation and transforming growth factor-ß-mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion.

SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival.

Platelet-derived SDF-1α (CXCL12) mediates inflammatory and regenerative mechanisms. The present study characterizes the effect of SDF-1α ligation in platelets. SDF-1α (0-100 µM) dose and time dependently caused internalization of its receptor CXCR4 (28.9 ± 1.6 vs. 16.1 ± 1.9 in SDF-1α-treated platelets), coupled to the surface externalization of CXCR7 (65.5 ± 8 vs. 162.8 ± 27.6 following SDF-1α treatment), both in vitro and in vivo. This was inhibited in the presence of AMD3100 (100 µM), CXCR4 blocking and vesicular transport inhibitors (brefeldin A, 10 µM; rapamycin, 100 nM). SDF-1α/CXCR-4-mediated CXCR7 translocation was significantly reduced by inhibitors of ERK1/2-(U0126-10 µM) and cyclophilinA (CyPA)-(NIM811-10 µM) by 28 and 46%, respectively. Further, SDF-1α-induced downstream phosphorylation of Erk1/2 led to CyPA-dependent ubiquitination of CXCR7, which is essential for its surface translocation. CyPA-PPIase-activity inhibitor NIM-811, Erk1/2, and E1-ligase inhibitor-(PYR-41-25 µM) significantly abolished SDF-1α-driven CXCR7 ubiquitination and subsequent surface translocation. SDF-1α induced CXCR7 ubiquitination, and its surface exposure was observed in wild-type murine platelets, but not in CyPA-deficient platelets. SDF-1α/CXCR4-CyPA-dependent CXCR7 translocation and its subsequent ligation attenuated activation-induced apoptosis both in vitro and when administered in vivo. This antiapoptotic effect of SDF-1α was abrogated by blocking CXCR7, also significantly affected in Cypa(-/-) platelets. Thus, we decipher a novel mechanism, whereby SDF-1α regulates relative receptor availability in circulating platelets and exerts its prosurvival benefits.-Chatterjee, M., Seizer, P., Borst, O., Schönberger, T., Mack, A., Geisler, T., Langer, H. F., May, A. E., Vogel, S., Lang, F., Gawaz, M. SDF-1α induces differential trafficking of CXCR4-CXCR7 involving cyclophilin A, CXCR7 ubiquitination and promotes platelet survival.

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus-mediated inflammatory cardiomyopathy.

In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis.

Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following ß-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.

Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation.

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes.

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)ß3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1µM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/ß and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10µM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

The inflammatory chemokine CXC motif ligand 16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling.

RATIONALE: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. OBJECTIVE: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. METHODS AND RESULTS: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin α(IIb)ß(3) activation, and shape change. CXCL16 increased Akt phosphorylation (Thr(308)/Ser(473)), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α(IIb)ß(3) activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000(-s)) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P(2)Y(1) (MRS2179, 100 µmol/L) and especially P(2)Y(12) (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. CONCLUSIONS: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6-dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Circulating platelet-progenitor cell coaggregate formation is increased in patients with acute coronary syndromes and augments recruitment of CD34+ cells in the ischaemic microcirculation.

AIMS: The aim of the present study was to evaluate the levels of platelet interaction with circulating CD34+ cells in patients with stable angina pectoris (SAP) and acute coronary syndromes (ACS) and to study the functional consequence of coaggregates formation in vitro and in vivo. METHODS AND RESULTS: Platelet binding to circulating progenitor cells was defined by the presence of the platelet-specific marker glycoprotein Ib (CD42b) on the surface of CD34+ cells using flow cytometry. The percentage of CD34+/CD42b+ cell coaggregates was increased in patients with ACS (n = 162), and especially in patients with ST-elevation myocardial infarction (STEMI) (n = 44), compared with patients with SAP (n = 116; P < 0.001). In the ANCOVA analysis, platelet/CD34+ cell coaggregates were independently increased in ACS after adjustment for possible confounders. In a subgroup of our cohort, we also evaluated the levels of CD34+/CD133+/CD42b+ cell coaggregates, which were also significantly increased in ACS, and especially in STEMI (P < 0.05). Platelet/CD34+ cell coaggregates formation correlated with platelet activation (P = 0.001). In a prospective pilot study of patients with AMI (n = 40) using cardiac MRI, patients with increased baseline platelet/CD34+ cell coaggregates presented with a less myocardial infarct size and better left ventricular function at a 3-month follow-up compared with patients with lower coaggregates (P < 0.05 for all). The adhesion of platelet/CD34+ cell coaggregates onto the extracellular matrix and to endothelial monolayer was enhanced compared with CD34+ under high shear rates in vitro (P < 0.05) and within the microcirculation in mice after ischaemia/reperfusion injury as assessed by intravital microscopy (P < 0.05). CONCLUSIONS: These findings imply that circulating platelet/CD34+ cell coaggregate levels are increased in ACS, especially in STEMI, which may be a novel mechanism of domiciliation of CD34+ progenitor cells to the injured microvasculature after acute myocardial infarction.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

Diverse potential of secretome from natural killer cells and monocyte-derived macrophages in activating stellate cells.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-ß1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

Engagement of αIIbß3 (GPIIb/IIIa) with ανß3 integrin mediates interaction of melanoma cells with platelets: a connection to hematogenous metastasis.

A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbß3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνß3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνß3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνß3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.