Metabolic effects of nuclear receptor activation in vivo after 28-day oral exposure to three endocrine-disrupting chemicals.

Environmental exposure to endocrine-disrupting chemicals (EDCs) can lead to metabolic disruption, resulting in metabolic complications including adiposity, dyslipidemia, hepatic lipid accumulation, and glucose intolerance. Hepatic nuclear receptor activation is one of the mechanisms mediating metabolic effects of EDCs. Here, we investigated the potential to use a repeated dose 28-day oral toxicity test for identification of EDCs with metabolic endpoints. Bisphenol A (BPA), pregnenolone-16α-carbonitrile (PCN), and perfluorooctanoic acid (PFOA) were used as reference compounds. Male and female wild-type C57BL/6 mice were orally exposed to 5, 50, and 500 µg/kg of BPA, 1000, 10 000, and 100 000 µg/kg of PCN and 50 and 300 µg/kg of PFOA for 28 days next to normal chow diet. Primary endpoints were glucose tolerance, hepatic lipid accumulation, and plasma lipids. After 28-day exposure, no changes in body weight and glucose tolerance were observed in BPA-, PCN-, or PFOA-treated males or females. PCN and PFOA at the highest dose in both sexes and BPA at the middle and high dose in males increased relative liver weight. PFOA reduced plasma triglycerides in males and females, and increased hepatic triglyceride content in males. PCN and PFOA induced hepatic expression of typical pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR)α target genes, respectively. Exposure to BPA resulted in limited gene expression changes. In conclusion, the observed changes on metabolic health parameters were modest, suggesting that a standard repeated dose 28-day oral toxicity test is not a sensitive method for the detection of the metabolic effect of EDCs.

Recent advances in novel therapies for lipid disorders.

The prevalence of lipid disorders is alarmingly increasing in the Western world. They are the result of either primary causes, such as unhealthy lifestyle choices or inherited risk factors, or secondary causes like other diseases or medication. Atypical changes in the synthesis, processing and catabolism of lipoprotein particles may lead to severe hypercholesterolemia, hypertriglyceridemia or elevated Lp(a). Although cholesterol-lowering drugs are the most prescribed medications, not all patients achieve guideline recommended cholesterol levels with the current treatment options, emphasising the need for new therapies. Also, some lipid disorders do not have any treatment options but rely only on stringent dietary restriction. Patients with untreated lipid disorders carry a severe risk of cardiovascular disease, diabetes, non-alcoholic fatty liver disease and pancreatitis among others. To achieve better treatment outcome, novel selective gene expression and epigenetic targeting therapies are constantly being developed. Therapeutic innovations employing targeted RNA technology utilise small interfering RNAs, antisense oligonucleotides, long non-coding RNAs and microRNAs to regulate target protein production whereas viral gene therapy provides functional therapeutic genes and CRISPR/Cas technology relies on gene editing and transcriptional regulation. In this review, we will discuss the latest advances in clinical trials for novel lipid-lowering therapies and potential new targets in pre-clinical phase.

The EDCMET Project: Metabolic Effects of Endocrine Disruptors.

Endocrine disruptors (EDs) are defined as chemicals that mimic, block, or interfere with hormones in the body's endocrine systems and have been associated with a diverse array of health issues. The concept of endocrine disruption has recently been extended to metabolic alterations that may result in diseases, such as obesity, diabetes, and fatty liver disease, and constitute an increasing health concern worldwide. However, while epidemiological and experimental data on the close association of EDs and adverse metabolic effects are mounting, predictive methods and models to evaluate the detailed mechanisms and pathways behind these observed effects are lacking, thus restricting the regulatory risk assessment of EDs. The EDCMET (Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways) project brings together systems toxicologists; experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive in vitro and in vivo methodological skills; and, ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. During its 5-year journey, EDCMET aims to identify novel ED mechanisms of action, to generate (pre)validated test methods to assess the metabolic effects of Eds, and to predict emergent adverse biological phenotypes by following the adverse outcome pathway (AOP) paradigm.

Deletion of Lymphangiogenic and Angiogenic Growth Factor VEGF-D Leads to Severe Hyperlipidemia and Delayed Clearance of Chylomicron Remnants.

Objective- Dyslipidemia is one of the key factors behind coronary heart disease. Blood and lymphatic vessels play pivotal roles in both lipoprotein metabolism and development of atherosclerotic plaques. Recent studies have linked members of VEGF (vascular endothelial growth factor) family to lipid metabolism, but the function of VEGF-D has remained unexplored. Here, we investigated how the deletion of VEGF-D affects lipid and lipoprotein metabolism in atherogenic LDLR-/- ApoB100/100 mice. Approach and Results- Deletion of VEGF-D (VEGF-D-/-LDLR-/-ApoB100/100) led to markedly elevated plasma cholesterol and triglyceride levels without an increase in atherogenesis. Size distribution and hepatic lipid uptake studies confirmed a delayed clearance of large chylomicron remnant particles that cannot easily penetrate through the vascular endothelium. Mechanistically, the inhibition of VEGF-D signaling significantly decreased the hepatic expression of SDC1 (syndecan 1), which is one of the main receptors for chylomicron remnant uptake when LDLR is absent. Immunohistochemical staining confirmed reduced expression of SDC1 in the sinusoidal surface of hepatocytes in VEGF-D deficient mice. Furthermore, hepatic RNA-sequencing revealed that VEGF-D is also an important regulator of genes related to lipid metabolism and inflammation. The lack of VEGF-D signaling via VEGFR3 (VEGF receptor 3) led to lowered expression of genes regulating triglyceride and cholesterol production, as well as downregulation of peroxisomal ß-oxidation pathway. Conclusions- These results demonstrate that VEGF-D, a powerful lymphangiogenic and angiogenic growth factor, is also a major regulator of chylomicron metabolism in mice.

Sleeping Beauty Transposon Vectors in Liver-directed Gene Delivery of LDLR and VLDLR for Gene Therapy of Familial Hypercholesterolemia.

Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.

Lymphatic vessel insufficiency in hypercholesterolemic mice alters lipoprotein levels and promotes atherogenesis.

OBJECTIVE: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. APPROACH AND RESULTS: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). CONCLUSIONS: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.

Lymphatic insufficiency leads to distinct myocardial infarct content assessed by magnetic resonance TRAFFn, T1ρ and T2 relaxation times.

The role of cardiac lymphatics in the pathogenesis of myocardial infarction (MI) is unclear. Lymphatic system regulates cardiac physiological processes such as edema and tissue fluid balance, which affect MI pathogenesis. Recently, MI and fibrosis have been assessed using endogenous contrast in magnetic resonance imaging (MRI) based on the relaxation along a fictitious field with rank n (RAFFn). We extended the RAFFn applications to evaluate the effects of lymphatic insufficiency on MI with comparison to longitudinal rotating frame (T1ρ) and T2 relaxation times. MI was induced in transgenic (TG) mice expressing soluble decoy VEGF receptor 3 that reduces lymphatic vessel formation and their wild-type (WT) control littermates for comparison. The RAFFn relaxation times with rank 2 (TRAFF2), and rank 4 (TRAFF4), T1ρ and T2 were acquired at time points 0, 3, 7, 21 and 42 days after the MI at 9.4 T. Infarct sizes were determined based on TRAFF2, TRAFF4, T1ρ and T2 relaxation time maps. The area of differences (AOD) was calculated based on the MI areas determined on T2 and TRAFF2, TRAFF4 or T1ρ relaxation time maps. Hematoxylin-eosin and Sirius red stained histology sections were prepared to confirm MI locations and sizes. MI was detected as increased TRAFF2, TRAFF4, T1ρ and T2 relaxation times. Infarct sizes were similar on all relaxation time maps during the experimental period. Significantly larger AOD values were found together with increased AOD values in the TG group compared to the WT group. Histology confirmed these findings. The lymphatic deficiency was found to increase cardiac edema in MI. The combination of TRAFF2 (or TRAFF4) and T2 characterizes MI and edema in the myocardium in both lymphatic insufficiency and normal mice without any contrast agents.

Downregulation of VEGFR3 signaling alters cardiac lymphatic vessel organization and leads to a higher mortality after acute myocardial infarction.

Heart has a wide lymphatic network but the importance of cardiac lymphatic system in heart diseases has remained unclear. Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) is a key molecule in the development and maintenance of cardiac lymphatic vessels. Here we characterized the role of VEGFR3 in healthy hearts and after myocardial infarction (MI) by using sVEGFR3 transgenic mice expressing a soluble decoy VEGFR3 under K14 promoter and Chy mice which have an inactivating mutation in the VEGFR3 gene. Cardiac lymphatic vessels were significantly dilated in the healthy hearts of sVEGFR3 mice when compared to controls. Lymphatic vessels formed large sheet-like structures in Chy mice. Attenuated VEGFR3 signaling led to a more severe MI predisposing to a significantly higher mortality in sVEGFR3 mice than in control mice. sVEGFR3 mice displayed intramyocardial hemorrhages in the infarcted area indicating hyperpermeability of the vasculature. Furthermore, novel MRI methods TRAFF2 and TRAFF4 and histological analysis revealed a modified structure of the fibrotic infarcted area in sVEGFR3 mice. In conclusion, the downregulation of VEGFR3 signaling modifies the structure of cardiac lymphatic network and causes vascular leakiness and increased mortality after MI.

Cardiac Lymphatics - A New Avenue for Therapeutics?

Recent progress in lymphatic vessel biology and in novel imaging techniques has established the importance of the lymphatic vasculature as part of the cardiovascular system. The lymphatic vessel network regulates many physiological processes important for heart function such as fluid balance, transport of extravasated proteins, and trafficking of immune cells. Therefore, lymphangiogenic therapy could be beneficial in the treatment of cardiovascular diseases, for example by improving reverse cholesterol transport (RCT) from atherosclerotic lesions or by resolving edema and fibrosis after myocardial infarction. In this review we first describe recent findings on the development and function of cardiac lymphatic vessels, and subsequently focus on the prospects of pro- and anti-lymphangiogenic therapies in cardiovascular diseases.

Soluble vascular endothelial growth factor receptor-1 improves therapeutic efficacy of suicide gene therapy in an angiogenesis-independent manner.

Abstract Malignant gliomas (MGs) are highly vascularized, aggressive brain cancers carrying a dismal prognosis. Because of their high vascularity, anti-angiogenic therapy is a potential treatment option. Indeed, the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has demonstrated promising results in clinical trials. Similarly, adenovirus-medicated Herpes simplex virus thymidine kinase and ganciclovir (AdHSV-tk/GCV) suicide gene therapy has established itself in clinical trials as a potential novel therapeutic strategy for MGs. In this study, we demonstrate the feasibility of combining adenovirus-mediated soluble VEGF receptor-1 anti-angiogenic gene therapy with AdHSV-tk/GCV suicide gene therapy to treat experimental MGs. Our results reveal that, apart from inhibiting angiogenesis, other anti-tumor mechanisms, such as reduction of infiltration by tumor-associated macrophages/microglia, may contribute to the improved therapeutic benefit of combination therapy.

Pro- and anti-angiogenic therapy and atherosclerosis with special emphasis on vascular endothelial growth factors.

INTRODUCTION: Atherosclerosis is a complex, progressive disease affecting nearly half of the population in Western countries. Although several treatment methods are already available, they may not be applicable to all patients suffering from advanced cardiovascular diseases, such as end-stage myocardial ischemia or difficult peripheral ischemia and potential new treatment methods are under intensive investigation. AREAS COVERED: VEGFs are major angiogenic molecules controlling vascular growth and function, vascular homeostasis, permeability and vasodilatation. Therefore, they have been regarded as potential new treatment agents for ischemic heart and peripheral vascular disease and several pro-angiogenic clinical trials have been conducted. In contrast, VEGFs also take part in pathological states by inducing microvessel growth in, for example tumors and atherosclerotic lesions. In this review, the biological basis of atherosclerosis and VEGF biology are presented as well as the latest results from pre-clinical research and clinical trials of pro- and anti-angiogenic therapy. EXPERT OPINION: Even though pro-angiogenesis has been shown to be safe and well-tolerated in clinical trials, efficacy of the treatment has not been satisfactory. In the expert opinion section of the review, we discuss the major obstacles to cardiovascular gene therapy and some future prospects.

Avidin fusion protein-expressing lentiviral vector for targeted drug delivery.

One of the main objectives of cancer therapy is to enhance the effectiveness of the drug by concentrating it at the target site and to minimize the undesired side effects to nontarget cells. We have previously constructed a fusion protein, Lodavin, consisting of avidin and the endocytotic part of the low-density lipoprotein receptor, and demonstrated its applicability to transient drug targeting in vivo. In this study we produced a lentiviral vector expressing this fusion protein and evaluated its safety and efficacy. The results showed that lentivirus-mediated gene transfer led to long-term avidin fusion protein expression on glioma cells and that the receptor was able to bind biotinylated compounds. Repeated administration was proven feasible and the optimal time frame(s) for administration of biotinylated therapeutic and/or imaging compounds was elucidated. Intravenous or intracranial injection of the virus into BDIX rats led to the production of antibodies against transgene (avidin), but repeated administration of the vector was unable to boost this effect. Neutralizing antibodies against the lentivirus were also detected. Furthermore, we showed that the anti-avidin antibodies did not significantly affect the ligand-binding capacity of the avidin fusion protein. The therapeutic efficacy of avidin fusion protein in tumor treatment was tested in vitro with biotinylated and nonbiotinylated nanoparticles loaded with paclitaxel. In vivo applicability of lentivirus was studied in the BDIX rat glioma model, in which high receptor expression was detected in the tumor area. The lentivirus-mediated delivery of the avidin fusion protein thus represents a potential approach for the repeated targeting of cytotoxic compounds to cancer cells.