Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

AimsThe antidiabetic drug metformin is currently used prior and during pregnancy for polycystic ovary syndrome, as well as during gestational diabetes mellitus. We investigated the effects of prenatal metformin exposure on the metabolic phenotype of the offspring during adulthood in mice.MethodsMetformin (300 mg/kg) or vehicle was administered orally to dams on regular diet from the embryonic day E0.5 to E17.5. Gene expression profiles in liver and brain were analysed from 4-day old offspring by microarray. Body weight development and several metabolic parameters of offspring were monitored both during regular diet (RD-phase) and high fat diet (HFD-phase). At the end of the study, two doses of metformin or vehicle were given acutely to mice at the age of 20 weeks, and Insig-1 and GLUT4 mRNA expressions in liver and fat tissue were analysed using qRT-PCR.ResultsMetformin exposed fetuses were lighter at E18.5. There was no effect of metformin on the maternal body weight development or food intake. Metformin exposed offspring gained more body weight and mesenteric fat during the HFD-phase. The male offspring also had impaired glucose tolerance and elevated fasting glucose during the HFD-phase. Moreover, the expression of GLUT4 mRNA was down-regulated in epididymal fat in male offspring prenatally exposed to metformin. Based on the microarray and subsequent qRT-PCR analyses, the expression of Insig-1 was changed in the liver of neonatal mice exposed to metformin prenatally. Furthermore, metformin up-regulated the expression of Insig-1 later in development. Gene set enrichment analysis based on preliminary microarray data identified several differentially enriched pathways both in control and metformin exposed mice.ConclusionsThe present study shows that prenatal metformin exposure causes long-term programming effects on the metabolic phenotype during high fat diet in mice. This should be taken into consideration when using metformin as a therapeutic agent during pregnancy.

AimsDespite the wide use of metformin in metabolically challenged pregnancies, the long-term effects on the metabolism of the offspring are not known. We studied the long-term effects of prenatal metformin exposure during metabolically challenged pregnancy in mice.Materials and MethodsFemale mice were on a high fat diet (HFD) prior to and during the gestation. Metformin was administered during gestation from E0.5 to E17.5. Male and female offspring were weaned to a regular diet (RD) and subjected to HFD at adulthood (10-11 weeks). Body weight and several metabolic parameters (e.g. body composition and glucose tolerance) were measured during the study. Microarray and subsequent pathway analyses on the liver and subcutaneous adipose tissue of the male offspring were performed at postnatal day 4 in a separate experiment.ResultsPrenatal metformin exposure changed the offspring's response to HFD. Metformin exposed offspring gained less body weight and adipose tissue during the HFD phase. Additionally, prenatal metformin exposure prevented HFD-induced impairment in glucose tolerance. Microarray and annotation analyses revealed metformin-induced changes in several metabolic pathways from which electron transport chain (ETC) was prominently affected both in the neonatal liver and adipose tissue.ConclusionThis study shows the beneficial effects of prenatal metformin exposure on the offspring's glucose tolerance and fat mass accumulation during HFD. The transcriptome data obtained at neonatal age indicates major effects on the genes involved in mitochondrial ATP production and adipocyte differentiation suggesting the mechanistic routes to improved metabolic phenotype at adulthood.

BACKGROUND AND PURPOSEThe objective of this study was to determine how the AMPK activating antidiabetic drug metformin affects the major activator of hepatic gluconeogenesis, PPARγ coactivator 1α (PGC-1α) and liver functions regulated by PGC-1α. EXPERIMENTAL APPROACHMouse and human primary hepatocytes and mice in vivo were treated with metformin. Adenoviral overexpression, siRNA and reporter gene constructs were used for mechanistic studies. KEY RESULTSMetformin increased PGC-1α mRNA and protein expression in mouse primary hepatocytes. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) (another AMPK activator) had the opposite effect. Metformin also increased PGC-1α in human primary hepatocytes; this effect of metformin was abolished by AMPK inhibitor compound C and sirtuin 1 siRNA. AMPK overexpression by AMPK-Ad also increased PGC-1α. Whereas metformin increased PGC-1α, it down-regulated gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Furthermore, metformin attenuated the increase in PEPCK and G6Pase mRNAs induced by PGC-1α overexpression, but did not affect PGC-1α-mediated induction of mitochondrial genes. Metformin down-regulated several key transcription factors that mediate the effect of PGC-1α on gluconeogenic genes including Krüppel-like factor 15, forkhead box protein O1 and hepatocyte NF 4α, whereas it increased nuclear respiratory factor 1, which is involved in PGC-1α-mediated regulation of mitochondrial proteins. CONCLUSIONS AND IMPLICATIONSDown-regulation of PGC-1α is not necessary for suppression of gluconeogenic genes by metformin. Importantly, metformin selectively affects hepatic PGC-1α-mediated gene regulation and prevents activation of gluconeogenesis, but does not influence its regulation of mitochondrial genes. These results identify selective modulation of hepatic PGC-1α functions as a novel mechanism involved in the therapeutic action of metformin. AbbreviationsACC, acetyl-CoA; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; ATP5β, ATP synthase subunit β; Cox, cytochrome c oxidase; Cox4i1, cytochrome c oxidase IV isoform 1; CRTC, cAMP response element-binding protein coactivator ; Cycs, cytochrome c; db-cAMP, dibutyryl-cAMP; FOXO1, forkhead box protein O1; G6Pase, glucose-6-phosphatase; HNF-4α, hepatocyte NF 4α; KLF15, Krüppel-like factor 15; LKB1, liver kinase B1; NAD, nicotinamide adenine dinucleotide; NRF1, nuclear respiratory factor 1; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1α, PPARγ coactivator 1α; SHP, small heterodimer partner NR0B2; SIRT1, sirtuin 1

An increasing amount of evidence indicates that a small extracellular chondroitin/dermatan sulfate proteoglycan, decorin, is indirectly involved in angiogenesis. Given that angiogenesis is a sine qua non for tumor growth and progression, we attempted to examine whether human malignant vascular tumors differ from human benign vascular tumors in terms of their decorin expression and synthesis. CD31 immunostaining demonstrated that the human malignant vascular tumors Kaposi's sarcoma and angiosarcoma were filled with capillary-like structures, whereas in benign cavernous and capillary hemangiomas, blood vessels were not as abundantly present. By utilizing in situ hybridization and immunocytochemical assays for decorin, we showed that there was no detectable decorin mRNA expression or immunoreactivity within the tumor mass in the Kaposi's sarcoma or angiosarcoma group. Instead, decorin was expressed in the connective tissue stroma lining the sarcoma tissue. In contrast to sarcomas, in hemangiomas, decorin mRNA expression and immunoreactivity were observed also within the tumor mass, particularly in the connective tissue stroma surrounding the clusters of intratumoral blood vessels. Finally, distribution of type I collagen was found to be similar to that of decorin in these tumor tissues. Our findings can be explained with different states of angiogenesis in dissimilar growths. In sarcomas, angiogenesis is extremely powerful, whereas in hemangiomas, angiogenesis has ceased. Thus, decorin is likely to possess a suppressive effect on human tumor angiogenesis in vivo, as previously described by studies using different experimental models. Decorin certainly provides a usable biomarker for distinguishing between benign and malignant vascular tumors in patients.

A therosclerosis is a chronic inflammatory disease causing progressive lesion formation and luminal narrowing of arteries. The underlying pathology is initiated by endothelial dysfunction and structural alterations after an imbalanced lipid metabolism and trapping of low-density lipoprotein (LDL) particles in the intima of arteries.1,2 Subsequently, intimal lipid accumulation triggers the release of various chemokines by activated endothelial cells, inducing a multiphase cascade of leukocyte infiltration and inflammatory responses in the arterial walls. As the knowledge of these inflammatory pathways has been growing during the past years, it has also uncovered new druggable targets that could complement the current treatment options, which do not directly interfere with the central inflammatory mechanisms driving atheroprogression. One endogenous pathway that has gained increasing attention for its wide-ranging and potent actions in suppressing maladaptive inflammatory responses is the melanocortin system consisting of melanocortin peptides and their cellular receptors. 3,4 However, the role of melanocortin signaling and its promise as a therapeutic target in atherosclerosis remain fully unexplored.Melanocortins are a family of peptides that are proteolytically cleaved from the common precursor molecule pro-opiomelanocortin. These peptides include melanocyte-stimulating hormones (α-, β-, and γ-MSH) and adrenocorticotropic hormone. They regulate important physiological functions by acting via G-protein-coupled melanocortin receptors (MC-Rs), named from MC1-R to MC5-R. 5,6 Our recent findings indicate that α-MSH serves as a protective regulator in the vasculature by enhancing endothelium-dependent control of blood vessel tone. 7 Mechanistically, α-MSH was shown to augment vascular nitric oxide (NO) availability by activating MC1-R in the endothelium. Besides promoting endothelial function, mounting evidence indicates that α-MSH, through the activation of © 2014 American Heart Association, Inc. Objective-Melanocortin peptides have been shown to elicit anti-inflammatory actions and to promote vascular endothelial function by activating type 1 and 3 melanocortin receptors. Here, we addressed whether these favorable properties of melanocortins could reduce atherosclerotic plaque inflammation and improve vasoreactivity in atherosclerotic mice. Approach and Results-Low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 were fed a high-fat diet for 8 or 16 weeks and treated with either vehicle or a stable melanocortin analog, melanotan II (MT-II, 0.3 mg/kg per day, 4 weeks). We determined plaque uptake of fluorine-18-labeled fluorodeoxyglucose as a surrogate marker for atherosclerotic plaque inflammation and vascular function of the aorta by ex vivo analyses. MT-II had no effect on body weight or composition, or plasma cholesterol levels in atherosclerotic mice. Without attenuating atherosclerotic lesion size or lesional macrophage accumulation, MT-II treatment reduced fluorine-18-labeled...

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