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Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells. Cancer Res; 72(23); 6227–35. ©2012 AACR.

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The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies. Electronic supplementary material The online version of this article (10.1007/s10456-020-09748-4) contains supplementary material, which is available to authorized users.

// Sanne A.M. van Lith 1, * , Ilse Roodink 1, 5, * , Joost J.C. Verhoeff 2 , Petri I. Makinen 3 , Jari P. Lappalainen 3 , Seppo Yla-Herttuala 3, 4 , Jos Raats 5 , Erwin van Wijk 6 , Ronald Roepman 7 , Stef J. Letteboer 7 , Kiek Verrijp 1 , William P.J. Leenders 1 1 Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands 2 Department of Radiotherapy, Amsterdam Medical Center, 1100 DD, Amsterdam, The Netherlands 3 Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland 4 Science Service Center and Gene Therapy Unit, Kuopio University Hospital, 70210 Kuopio, Finland 5 Modiquest BV, LSP, Molenstraat 110, 5342 CC, Oss, The Netherlands 6 Department of Otorhinolaryngology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands 7 Department of Genetics, RadboudUMC, 6500 HB, Nijmegen,The Netherlands * These authors contributed equally to this work Correspondence to: William Leenders, email: william.leenders@radboudumc.nl Keywords: glioma, stroma, targeting, nanobody, macrophages Received: June 30, 2016 Accepted: September 19, 2016 Published: September 26, 2016 ABSTRACT Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs). Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150 Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150 Glued is a marker for activated endothelial cells. In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150 Glued as a novel targetable protein on activated endothelial cells and macrophages.

Aims Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR−/−), and LDLR−/− mice expressing apoB-100 only (LDLR−/− ApoB100/100) having a humanized lipoprotein profile. Methods and results LDLR−/− mice were fed a high-fat diet (HFD) for 6 or 12 weeks and LDLR−/−ApoB100/100 mice a regular chow diet for 6 or 12 months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR−/− mice reduced total plasma cholesterol after 6 weeks of HFD and triglycerides in LDLR−/−ApoB100/100 mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR−/−ApoB100/100 mice as it increased plaque calcification. Moreover, ∼36% of Nrf2−/−LDLR−/−ApoB100/100 females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12 months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR−/−ApoB100/100 female mice at age of 12 months. Conclusions Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR−/−ApoB100/100 mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.

Objective Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis. Methods Hypercholesterolemic (low-density lipoprotein receptor−/− apolipoprotein B100/100) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4–8.To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics. Results Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (−72%, p, Highlights • Antibody-mediated Ang-2 blockage delays fatty streak formation in mice. • Ang-2 blockage lowers plasma triglyceride levels. • Ang-2 blockage has no negative effects on preexisting atherosclerosis.

Aims The loss of nuclear factor E2-related factor 2 (Nrf2) has been shown to protect against atherogenesis in apoE-deficient mice. The mechanism by which Nrf2 deficiency affords atheroprotection in this model is currently unknown, but combined systemic and local vascular effects on lesion macrophages have been proposed. We investigated the effect of bone marrow-specific loss of Nrf2 on early atherogenesis in low-density lipoprotein (LDL) receptor-deficient (LDLR−/−) mice, and assessed the effect of Nrf2 on cellular accumulation of modified LDLs and the expression of inflammatory markers in macrophages. Methods and results The effect of bone marrow-specific loss of Nrf2 on atherogenesis was studied using bone marrow transplantation of wild-type (WT) or Nrf2−/− bone marrow to LDLR−/− mice. Mice transplanted with Nrf2−/− bone marrow and fed a high-fat diet for 6 weeks exhibited significantly larger atherosclerotic lesions than WT bone marrow transplanted mice. Moreover, in thioglycollate-elicited Nrf2−/− macrophages, the uptake of acetylated and malondialdehyde-modified LDLs was increased in comparison with WT controls, with the concomitant increase in the expression of scavenger receptor A and toll-like receptor 4. In addition, the expression of pro-inflammatory monocyte chemoattractant protein-1 and interleukin-6 were increased in Nrf2−/− vs. WT macrophages. Conclusion Nrf2 deficiency specific to bone marrow-derived cells aggravates atherosclerosis in LDLR−/− mice. Furthermore, the loss of Nrf2 in macrophages enhances foam cell formation and promotes the pro-inflammatory phenotype.

Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model.By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing similar to 53% of all VEGF-regulated transcripts and similar to 26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN.Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells. Cancer Res; 72(23); 6227–35. ©2012 AACR.

Background: Macrophage scavenger receptors SR-A and CD36 play a key role in the pathogenesis of atherosclerosis by participating in the uptake of modified lipoproteins, further leading to formation of foam cells. We have recently shown that RNAi-mediated silencing of these receptors decreases atherogenesis in hyperlipidemic LDLR -/- /ApoB 100/100 mice. Additionally, we discovered that there is a reciprocal regulation mechanism between these two receptors in mouse macrophages. Because there is limited information on the involvement of transcription factor NF-κB to SR-A and CD36 expression, the aim of this study was to investigate the role of NF-κB in the regulation of these receptors in detail. Methods and Results: Lentiviral vector mediated silencing of SR-A and CD36 alters the behavior of transcription factor NF-κB in mouse macrophages. After LPS exposure, SR-A silencing enhanced the classical NF-κB activation measured by activation of NF-κB responsive genes such as M-CSF, MCP-1 and ICAM-1. Moreover, CD36 silencing increased the expression of NF-κB subunit p50 with and without LPS stimulation by 2.7-fold (p Conclusion: These results suggest that SR-A and CD36 modulate macrophage functions under inflammatory conditions, and imply that SR-A and CD36 are important signal transducers in inflammatory events involving NF-κB.

Accumulation of macrophages, red blood cells and lipids in atherosclerotic plaques is associated with plaque rupture. It has been shown that macrophages and erythrocytes can enter the plaque also via plaque microvasculature originating from the adventitia. Furthermore, it has been reported that in advanced or ruptured human coronary lesions the microvessel density is increased and these microvessels are leaky. The leaky phenotype is characterized by poor pericyte coverage and dysfunctional inter-endothelial junctions. As angiopoietins are involved in angiogenic growth and maturation, we hypothesized that periadventitial Ang-1 or Ang-2 gene transfer would reduce atherogenesis and affect microvasculature in mouse carotid artery. To study this, atherosclerosis was induced by placing a perivascular silastic collar around the carotid artery of LDLr -/- ApoB100/100 mice fed a high cholesterol (0,15 %) diet for 3 weeks. Simultaneous to surgery adenoviral gene transfer (5μl, 5 x 107 pfu/ml) of either Ang-1, Ang-2 or LacZ as a control was applied to the carotid artery (20 mice per group). After 1 or 5 weeks follow-up on high cholesterol diet mice were sacrificed. Plaque size and microvessel density were analyzed with morphometry software (Leica Qwin) using cross-sections stained with hematoxylin and eosin or CD31 respectively. Microvessel ultrastructure was studied using electron microscopy. Ang-2 gene transfer led to significantly smaller plaque area and plaque volume 5 weeks after collar placement compared to LacZ group (2,7 fold, p In conclusion: Ang-2 over expression in mouse carotid arteries led to smaller plaque size and lower microvessel density, suggesting the involvement of angiopoietin signaling in angiogenesis of the atherosclerotic vessel wall.

Aims Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far, the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing, which is a clinically feasible method to down-regulate the expression of these receptors. Methods and results We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages, whereas the uptake of oxidized LDL was not altered. More importantly, silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR−/−ApoB100/100) mice kept on a western diet, silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%, respectively) in cross-sectional lesion area, whereas simultaneous silencing of both receptors was not effective. Conclusion Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However, due to reciprocal upregulation, silencing of both SR-A and CD36 is not effective.