Solid tumor therapy by selectively targeting stromal endothelial cells

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.

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Chrysin Suppresses Vascular Endothelial Inflammation via Inhibiting the NF-κB Signaling Pathway

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248418810809 ◽

2018 ◽

Vol 24 (3) ◽

pp. 278-287 ◽

Cited By ~ 4

Author(s):

Shengnan Zhao ◽

Minglu Liang ◽

Yilong Wang ◽

Ji Hu ◽

Yi Zhong ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Signaling Pathway ◽

Adhesion Molecule ◽

Intercellular Adhesion Molecule ◽

Flavonoid Compound ◽

Endothelial Inflammation ◽

Wide Range

The vascular endothelium is a continuous layer of flat polygonal cells that are in direct contact with the blood and participate in responses to inflammation. Chrysin is a flavonoid compound extracted from plants of the genus Asteraceae with a wide range of pharmacological activities and physiological activities. Here, we studied the effects of chrysin on the regulation of the proadhesion and pro-inflammatory phenotypes of the endothelium both in vitro and in vivo. Our results revealed that chrysin strongly inhibited Tohoku Hospital Pediatrics-1 (THP-1) cell adhesion to primary human umbilical vein endothelial cells and concentration-dependently attenuated interleukin 1β-induced increases in intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin messenger RNA levels and ICAM-1 and VCAM-1 protein levels. Previous studies reported that nuclear factor κB (NF-κB) is important in the inflammatory response in endothelial cells, particularly in regulating adhesion molecules, and our data shed light on the mechanisms whereby chrysin suppressed endothelial inflammation via the NF-κB signaling pathway. In addition, our in vivo findings demonstrated the effects of chrysin in the permeability and inflammatory responses of the endothelium to inflammatory injury. Taken together, we conclude that chrysin inhibits endothelial inflammation both in vitro and in vivo, which could be mainly due to its inhibition of NF-κB signaling activation. In conclusion, chrysin may serve as a promising therapeutic candidate for inflammatory vascular diseases.

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The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1510495112 ◽

2015 ◽

Vol 112 (46) ◽

pp. 14284-14289 ◽

Cited By ~ 35

Author(s):

Xavier Cullere ◽

Eva Plovie ◽

Paul M. Bennett ◽

Calum A. MacRae ◽

Tanya N. Mayadas

Keyword(s):

Endothelial Cells ◽

Cavernous Malformation ◽

Body Axis ◽

Cerebral Cavernous Malformations ◽

Loss Of Function ◽

Cavernous Malformations ◽

Downstream Target ◽

Density Flow

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe “big heart” and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

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Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200610086 ◽

2007 ◽

Vol 177 (3) ◽

pp. 539-549 ◽

Cited By ~ 85

Author(s):

Mark M. Fuster ◽

Lianchun Wang ◽

Janice Castagnola ◽

Lyudmila Sikora ◽

Krisanavane Reddi ◽

...

Keyword(s):

Endothelial Cells ◽

Heparan Sulfate ◽

Tumor Angiogenesis ◽

Wound Repair ◽

Reproductive Capacity ◽

Erk Phosphorylation ◽

Tumor Endothelium ◽

A Cell

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

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RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation ◽

10.1161/circulationaha.118.038379 ◽

2020 ◽

Author(s):

Denuja Karunakaran ◽

My-Anh Nguyen ◽

Michele Geoffrion ◽

Dianne Vreeken ◽

Zachary Lister ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Activation ◽

Inflammatory Responses ◽

Early Stage ◽

Oxidized Ldl ◽

Loss Of Function ◽

Aortic Sinus ◽

Atherosclerotic Lesions

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

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The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21145148 ◽

2020 ◽

Vol 21 (14) ◽

pp. 5148

Author(s):

Rawnaq Esa ◽

Eliana Steinberg ◽

Dvir Dror ◽

Ouri Schwob ◽

Mehrdad Khajavi ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Function ◽

Loss Of Function ◽

Methionine Aminopeptidase ◽

Zebrafish Model ◽

Lymphatic Capillary ◽

Intracellular Enzyme

During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.

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Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT inEscherichia coli

Journal of Biomedicine and Biotechnology ◽

10.1155/2008/564969 ◽

2008 ◽

Vol 2008 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Jiping Cai ◽

Ruili Wei ◽

Jinwei Cheng

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Recombinant Expression ◽

Tumor Therapy ◽

Vascular Endothelial Cell ◽

Chimeric Protein ◽

Growth Inhibitor ◽

Endothelial Cell Proliferation

Vascular endothelial cell growth inhibitor (VEGI) is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC) has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies inE. coliBL21 (DE3), and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

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Thermochemical Ablation: A Novel Technique for Solid Tumor Therapy

Journal of Medical Devices ◽

10.1115/1.3147268 ◽

2009 ◽

Vol 3 (2) ◽

Cited By ~ 1

Author(s):

M. Shenoi ◽

E. Cressman

Keyword(s):

Solid Tumors ◽

Tumor Tissue ◽

Tumor Therapy ◽

Exothermic Reaction ◽

Palliative Therapy ◽

Distal Portion ◽

Tissue Destruction ◽

Prototype Device

To overcome the limitations of existing ablation techniques, we propose a novel combinatorial approach that would utilize the thermal and chemical destructive effects of exothermic chemical reactions, such as an acid/base neutralization reaction, to treat solid tumors. Thermochemical ablation is a potential technique for percutaneous probe-based tumor therapy. It involves simultaneous intratumoral delivery of multiple reagents resulting in thermal energy released by an exothermic reaction to ablate tumor tissue with concurrent generation of a hyperosmolar byproduct that could accentuate tumor destruction. Besides the benefit of synergistic thermal and chemical effects for tumor tissue destruction, this technique is potentially highly cost-effective, easy to implement, and able to treat larger sized tumors. Our hypothesis is that thermochemical ablation can create an evenly distributed zone of coagulation in tumor tissue without systemic toxicity. A prototype device assembled using off-the-shelf components is being investigated in our lab for concurrent intraparenchymal delivery of an acid and a base. The distal portion of the multi-lumen device allows for passive mixing of the reagents before entering the tissue. The prototype device also satisfies other desirable design criteria such as rigidity to penetrate body tissue, reduced diameter, chemical stability to reagents, etc. However, the device can be improved upon by incorporating additional characteristics such as optimized imaging characteristic for real-time visualization and localization within tumor tissue, MRI compatibility, thermal insulation, improved mixing at the tip, etc. Our lab is currently working on improving the design of the infusion device as well as assessing the feasibility of the thermochemical ablation technique in vitro and in vivo. While currently being targeted conservatively for palliative therapy of unresectable or late-stage aggressive malignancies such as hepatocellular carcinoma, thermochemical ablation has potential use in the therapy of a majority of solid tumors such as breast cancer, lung cancer, prostate cancer, renal cancer, sarcomas, etc.

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miRNA-1246 in Extracellular Vesicles Secreted from Metastatic Tumor Induces Drug Resistance in Tumor Endothelial Cells

10.21203/rs.3.rs-60198/v1 ◽

2020 ◽

Author(s):

Chisaho Torii ◽

Nako Maishi ◽

Taisuke Kawamoto ◽

Masahiro Morimoto ◽

Kosuke Akiyama ◽

...

Keyword(s):

Drug Resistance ◽

Endothelial Cells ◽

Induced Resistance ◽

Extracellular Vesicles ◽

Metastatic Tumor ◽

Metastatic Tumors ◽

Glycoprotein P ◽

Tumor Endothelial Cells

Abstract Background: Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of p-glycoprotein (p-gp, ABCB1), contrary to traditional assumptions. Furthermore, p-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential p-gp expression in TECs remains unclear. Methods: miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. Results: In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on extracellular vesicles (EVs) and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. Conclusions: These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.

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Mitogen-Inducible Gene 6 Inhibits Angiogenesis by Binding to SHC1 and Suppressing Its Phosphorylation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.634242 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lixian Liu ◽

Liying Xing ◽

Rongyuan Chen ◽

Jianing Zhang ◽

Yuye Huang ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Adaptor Protein ◽

Model Systems ◽

Vascular Endothelial ◽

Loss Of Function ◽

Angiogenic Effect ◽

Inducible Gene

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.

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Prolyl 3-Hydroxylase 2 Is a Molecular Player of Angiogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22083896 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3896

Author(s):

Paola Pignata ◽

Ivana Apicella ◽

Valeria Cicatiello ◽

Caterina Puglisi ◽

Sara Magliacane Magliacane Trotta ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Collagen Iv ◽

Mitogen Activated Protein Kinase ◽

Vegf Receptor ◽

Loss Of Function ◽

Type Iv ◽

Umbilical Vein Endothelial Cells

Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3h2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.

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