The BCR::ABL1 tyrosine kinase inhibitors ponatinib and nilotinib differentially affect endothelial angiogenesis and signalling.

BCR::ABL1 inhibitors, the treatment of choice for the majority of patients with chronic myeloid leukaemia (CML), can cause vascular side effects that vary between agents. The exact underlying mechanisms are still poorly understood, but the vascular endothelium has been proposed as a site of origin. The present study investigates the effects of three BCR::ABL1 inhibitors, ponatinib, nilotinib and imatinib, on angiogenesis and signalling in human endothelial cells in response to vascular endothelial growth factor (VEGF). The experiments were performed in endothelial cells isolated from human umbilical veins. After exposure to imatinib, ponatinib and nilotinib, the angiogenic capacity of endothelial cells was assessed in spheroid assays. VEGF-induced signalling pathways were examined in Western blotting experiments using different specific antibodies. RNAi technology was used to downregulate proteins of interest. Intracellular cGMP levels were measured by ELISA. Imatinib had no effect on endothelial function. Ponatinib inhibited VEGF-induced sprouting, while nilotinib increased spontaneous and VEGF-stimulated angiogenesis. These effects did not involve wild-type ABL1 or ABL2, as siRNA-mediated knockdown of these kinases did not affect angiogenesis and VEGF signalling. Consistent with their effects on sprouting, ponatinib and nilotinib affected angiogenic pathways in opposite directions. While ponatinib inhibited VEGF-induced signalling and cGMP formation, nilotinib activated angiogenic signalling, in particular phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2). The latter occurred in an epidermal growth factor receptor (EGFR)-dependent manner possibly via suppressing Fyn-related kinase (FRK), a negative regulator of EGFR signalling. Both, pharmacological inhibition of Erk1/2 or EGFR suppressed nilotinib-induced angiogenic sprouting. These results support the notion that the vascular endothelium is a site of action of BCR::ABL1 inhibitors from which side effects may arise, and that the different vascular toxicity profiles of BCR::ABL1 inhibitors may be due to their different actions at the molecular level. In addition, the as yet unknown pro-angiogenic effect of nilotinib should be considered in the treatment of patients with comorbidities associated with pathological angiogenesis, such as ocular disease, arthritis or obesity.

Hyperthermia Influences the Secretion Signature of Tumor Cells and Affects Endothelial Cell Sprouting.

Tumors are a highly heterogeneous mass of tissue showing distinct therapy responses. In particular, the therapeutic outcome of tumor hyperthermia treatments has been inconsistent, presumably due to tumor versus endothelial cell cross-talks related to the treatment temperature and the tumor tissue environment. Here, we investigated the impact of the average or strong hyperthermic treatment (43 °C or 47 °C for 1 h) of the human pancreatic adenocarcinoma cell line (PANC-1 and BxPC-3) on endothelial cells (HUVECs) under post-treatment normoxic or hypoxic conditions. Immediately after the hyperthermia treatment, the distinct repression of secreted pro-angiogenic factors (e.g., VEGF, PDGF-AA, PDGF-BB, M-CSF), intracellular HIF-1α and the enhanced phosphorylation of ERK1/2 in tumor cells were detectable (particularly for strong hyperthermia, 2D cell monolayers). Notably, there was a significant increase in endothelial sprouting when 3D self-organized pancreatic cancer cells were treated with strong hyperthermia and the post-treatment conditions were hypoxic. Interestingly, for the used treatment temperatures, the intracellular HIF-1α accumulation in tumor cells seems to play a role in MAPK/ERK activation and mediator secretion (e.g., VEGF, PDGF-AA, Angiopoietin-2), as shown by inhibition experiments. Taken together, the hyperthermia of pancreatic adenocarcinoma cells in vitro impacts endothelial cells under defined environmental conditions (cell-to-cell contact, oxygen status, treatment temperature), whereby HIF-1α and VEGF secretion play a role in a complex context. Our observations could be exploited for the hyperthermic treatment of pancreatic cancer in the future.

The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis.

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3-regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes-platinum resistance and angiogenesis.

Circulating Bile Acids in Liver Failure Activate TGR5 and Induce Monocyte Dysfunction.

BACKGROUND & AIMS: Retention of bile acids in the blood is a hallmark of liver failure. Recent studies have shown that increased serum bile acid levels correlate with bacterial infection and increased mortality. However, the mechanisms by which circulating bile acids influence patient outcomes still are elusive. METHODS: Serum bile acid profiles in 33 critically ill patients with liver failure and their effects on Takeda G-protein-coupled receptor 5 (TGR5), an immunomodulatory receptor that is highly expressed in monocytes, were analyzed using tandem mass spectrometry, novel highly sensitive TGR5 bioluminescence resonance energy transfer using nanoluciferase (NanoBRET, Promega Corp, Madison, WI) technology, and in vitro assays with human monocytes. RESULTS: Twenty-two patients (67%) had serum bile acids that led to distinct TGR5 activation. These TGR5-activating serum bile acids severely compromised monocyte function. The release of proinflammatory cytokines (eg, tumor necrosis factor α or interleukin 6) in response to bacterial challenge was reduced significantly if monocytes were incubated with TGR5-activating serum bile acids from patients with liver failure. By contrast, serum bile acids from healthy volunteers did not influence cytokine release. Monocytes that did not express TGR5 were protected from the bile acid effects. TGR5-activating serum bile acids were a risk factor for a fatal outcome in patients with liver failure, independent of disease severity. CONCLUSIONS: Depending on their composition and quantity, serum bile acids in liver failure activate TGR5. TGR5 activation leads to monocyte dysfunction and correlates with mortality, independent of disease activity. This indicates an active role of TGR5 in liver failure. Therefore, TGR5 and bile acid metabolism might be promising targets for the treatment of immune dysfunction in liver failure.

Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

Inflammatory Conditions Disrupt Constitutive Endothelial Cell Barrier Stabilization by Alleviating Autonomous Secretion of Sphingosine 1-Phosphate.

The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.

VEGF Triggers Transient Induction of Autophagy in Endothelial Cells via AMPKα1.

AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner. We show that functional autophagy is required for VEGF-induced angiogenesis and may have specific functions in addition to maintaining homeostasis. In line with this, inhibition of autophagy impaired VEGF-mediated formation of the Notch intracellular domain, a critical regulator of angiogenesis. Our study characterizes autophagy induction as a pro-angiogenic function of the VEGF/AMPK pathway and suggests that timely activation of autophagy-initiating pathways may help to initiate angiogenesis.

Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model.

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

Hyperglycemia-induced endothelial dysfunction is alleviated by thioredoxin mimetic peptides through the restoration of VEGFR-2-induced responses and improved cell survival.

BACKGROUND: Diabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. METHODS AND RESULTS: Human umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of "VEGF resistance" phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. CONCLUSIONS: Our findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.

The Peroxisome Proliferator-Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

Phenotypic and functional differences between senescent and aged murine microglia.

Microglia, the key innate immune cells in the brain, have been reported to drive brain aging and neurodegenerative disorders; however, few studies have analyzed microglial senescence and the impact of aging on the properties of microglia. In the present study, we characterized senescence- and aging-associated phenotypes of murine brain microglia using well-accepted markers, including telomere length, telomerase activity, expression of p16INK4a, p21, p53, senescence-associated ß-galactosidase, and a senescence-associated secretory phenotype. Quantitative real-time polymerase chain reaction analysis and a Telomeric Repeat Amplification Protocol assay indicated shortened telomeres and increased telomerase activity in senescent microglia, whereas telomeres remained unaltered and telomerase activity was reduced in aged microglia. Senescent microglia upregulated p16INK4a, p21, and p53, whereas acutely isolated microglia from the aged brain only exhibited a modest upregulation of p16INK4a. Senescent microglia showed decreased proliferation, while it was unchanged in aged microglia. Furthermore, microglia at late passages strongly upregulated expression of the senescent marker senescence-associated ß-galactosidase. Senescent and aged microglia exhibited differential activation profiles and altered responses to stimulation. We conclude that microglia from the aged mouse brain do not show typical senescent changes because their phenotype and functional response strongly differ from those of senescent microglia in vitro.

Candida albicans ß-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages.

ß-Glucan derived from cell walls of Candida albicans is a potent immune modulator. It has been shown to induce trained immunity in monocytes via epigenetic and metabolic reprogramming and to protect from lethal sepsis if applied prior to infection. Since ß-glucan-trained monocytes have not been classified within the system of mononuclear phagocytes we analyzed these cells metabolically, phenotypically and functionally with a focus on monocyte-to-macrophage differentiation and compared them with naïve monocytes and other types of monocyte-derived cells such as classically (M1) or alternatively (M2) activated macrophages and monocyte-derived dendritic cells (moDCs). We show that ß-glucan inhibits spontaneous apoptosis of monocytes independent from autocrine or paracrine M-CSF release and stimulates monocyte differentiation into macrophages. ß-Glucan-differentiated macrophages exhibit increased cell size and granularity and enhanced metabolic activity when compared to naïve monocytes. Although ß-glucan-primed cells expressed markers of alternative activation and secreted higher levels of IL-10 after lipopolysaccharide (LPS), their capability to release pro-inflammatory cytokines and to kill bacteria was unaffected. Our data demonstrate that ß-glucan priming induces a population of immune competent long-lived monocyte-derived macrophages that may be involved in immunoregulatory processes.

GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Phosphoinositide 3-kinase gamma controls inflammation-induced myocardial depression via sequential cAMP and iNOS signalling.

AIMS: Sepsis-induced myocardial depression (SIMD), an early and frequent event of infection-induced systemic inflammatory response syndrome (SIRS), is characterized by reduced contractility irrespective of enhanced adrenergic stimulation. Phosphoinositide-3 kinase γ (PI3Kγ) is known to prevent ß-adrenergic overstimulation via its scaffold function by activating major cardiac phosphodiesterases and restricting cAMP levels. However, the role of PI3Kγ in SIRS-induced myocardial depression is unknown. This study is aimed at determining the specific role of lipid kinase-dependent and -independent functions of PI3Kγ in the pathogenesis of SIRS-induced myocardial depression. METHODS AND RESULTS: PI3Kγ knockout mice (PI3Kγ(-/-)), mice expressing catalytically inactive PI3Kγ (PI3Kγ(KD/KD)), and wild-type mice (P3Kγ(+/+)) were exposed to lipopolysaccharide (LPS)-induced systemic inflammation and assessed for survival, cardiac autonomic nervous system function, and left ventricular performance. Additionally, primary adult cardiomyocytes were used to analyse PI3Kγ effects on myocardial contractility and inflammatory response. SIRS-induced adrenergic overstimulation induced a transient hypercontractility state in PI3Kγ(-/-) mice, followed by reduced contractility. In contrast, P3Kγ(+/+) mice and PI3Kγ(KD/KD) mice developed an early and ongoing myocardial depression despite exposure to similarly increased catecholamine levels. Compared with cells from P3Kγ(+/+) and PI3Kγ(KD/KD) mice, cardiomyocytes from PI3Kγ(-/-) mice showed an enhanced and prolonged cAMP-mediated signalling upon norepinephrine and an intensified LPS-induced proinflammatory response characterized by nuclear factor of activated T-cells-mediated inducible nitric oxide synthase up-regulation. CONCLUSIONS: This study reveals the lipid kinase-independent scaffold function of PI3Kγ as a mediator of SIMD during inflammation-induced SIRS. Activation of cardiac phosphodiesterases via PI3Kγ is shown to restrict myocardial hypercontractility early after SIRS induction as well as the subsequent inflammatory responses.

Malarial pigment hemozoin impairs chemotactic motility and transendothelial migration of monocytes via 4-hydroxynonenal.

Natural hemozoin, nHZ, is avidly phagocytosed in vivo and in vitro by human monocytes. The persistence of the undigested ß-hematin core of nHZ in the phagocyte lysosome for long periods of time modifies several cellular immune functions. Here we show that nHZ phagocytosis by human primary monocytes severely impaired their chemotactic motility toward MCP-1, TNF, and FMLP, by approximately 80% each, and their diapedesis across a confluent human umbilical vein endothelial cell layer toward MCP-1 by 45±5%. No inhibition was observed with latex-fed or unfed monocytes. Microscopic inspection revealed polarization defects in nHZ-fed monocytes due to irregular actin polymerization. Phagocytosed nHZ catalyzes the peroxidation of polyunsaturated fatty acids and generation of the highly reactive derivative 4-hydroxynonenal (4-HNE). Similar to nHZ phagocytosis, the exposure of monocytes to in vivo-compatible 4-HNE concentrations inhibited cell motility in both the presence and the absence of chemotactic stimuli, suggesting severe impairment of cytoskeleton dynamics. Consequently, 4-HNE conjugates with the cytoskeleton proteins ß-actin and coronin-1A were immunochemically identified in nHZ-fed monocytes and mass spectrometrically localized in domains of protein-protein interactions involved in cytoskeleton reorganization and cell motility. The molecular and functional modifications of actin and coronin by nHZ/4-HNE may also explain impaired phagocytosis, another motility-dependent process previously described in nHZ-fed monocytes. Further studies will show whether impaired monocyte motility may contribute to the immunodepression and the frequent occurrence of secondary infections observed in malaria patients.

Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery.

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(®). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.

NOS inhibition synchronizes calcium oscillations in human adipose tissue-derived mesenchymal stem cells by increasing gap-junctional coupling.

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.

Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independently of nitric-oxide synthase.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state and a regulator of cellular homeostasis. In endothelial cells, AMPK is stimulated via the upstream kinases LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Previously, AMPK has been reported to activate endothelial nitric-oxide synthase (eNOS). Using genetic and pharmacological approaches, we show that vascular endothelial growth factor (VEGF) stimulates AMPK in human and mice endothelial cells via CaMKKbeta. VEGF-induced AMPK activation is potentiated under conditions of energy deprivation induced by 2-deoxyglucose. To investigate the role of AMPK in endothelial function, CaMKKbeta, AMPKalpha1, or AMPKalpha2 was down-regulated by RNA interference, and studies in AMPKalpha1(-/-) mice were performed. We demonstrate that AMPK does not mediate eNOS phosphorylation at serine residue 1177 or 633, NO- dependent cGMP generation, or Akt phosphorylation in response to VEGF. Using inhibitors of eNOS or soluble guanylyl cyclase and small interfering RNA against eNOS, we show that NO does not act upstream of AMPK. Taken together, these data indicate that VEGF-stimulated AMPK and eNOS pathways act independently of each other. However, acetyl-CoA carboxylase, a key enzyme in the regulation of fatty acid oxidation, was phosphorylated in response to VEGF in an AMPKalpha1- and AMPKalpha2-dependent manner. Our results show that AMPKalpha1 plays an essential role in VEGF-induced angiogenesis in vitro (tube formation and sprouting from spheroids) and in vivo (Matrigel plug assay). In contrast, AMPKalpha2 was not involved in VEGF-triggered sprouting. The data suggest that AMPKalpha1 promotes VEGF-induced angiogenesis independently of eNOS, possibly by providing energy via inhibition of acetyl-CoA carboxylase.