SVEC4-10 Cells Research Articles

Unlocking the potential of biocompatible chitosan-hyaluronic acid nanogels labeled with fluorochromes: A promising step toward enhanced FRET bioimaging.

Chitosan is a natural polysaccharide widely used in medical formulations as nanoparticles due to their special properties. Our work aimed to assess the biocompatibility of chitosan-hyaluronic acid nanogels labeled with fluorochromes for use in biomedical applications, based on the FRET effect. The preparation method included the ionic gelation, grafting rhodamine or fluorescein isothiocyanate molecules onto the chitosan backbone. To assess the potential applications as fluorescence imaging tools of chitosan-fluorophores conjugates in diagnostics and therapies, SVEC4-10 cells (simian virus 40-transformed mouse microvascular endothelial cell line) and RAW264.7 murine macrophages were used within this study. Good biocompatibility was observed after 6 and 24 h of incubation with nanogels, with no increase in cell death or membrane damage for concentrations up to 120 μg/mL. Both types of fluorescent nanogels presented the tendency to agglomerate on the cell membrane's surface, and few cells were internalized, especially at the periphery of cells. Molecular dynamics simulations showed that distances between fluorophores fitted at values close to those calculated based on FRET experiments. These formulations can further incorporate gadolinium for better nanomedicine tools.

Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene.

Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene isolated from plant essential oils, such as tea tree (Melaleuca alternifolia), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris Marchand), and eucalyptus (Eucalyptus sp.). Terpenes are widely studied molecules pharmacologically active on the cardiovascular system, hemostasis, and antioxidant actions. Herein, it was investigated the cytotoxic and antiplatelet activity of γ-TPN using different non-clinical laboratory models. For in silico evaluation, the PreADMET, SwissADME, and SwissTargetPrediction softwares were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay upon normal murine endothelial SVEC4-10 and fibroblast L-929 cells. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100, and 200 µM) for 24 h. SHR animals were pre-treated by gavage with γ-TPN for 7 days and divided into four groups (negative control, 25, 50, and 100 mg/kg). Blood samples were collected to measure nitrite using the Griess reagent. Gamma-TPN proved to be quite lipid-soluble (Log P = +4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. It exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L-929 (CC50 = 333.3 µM) and SVEC 4-10 (CC50 = 366.7 µM)cells. The presence of γ-TPN in SVEC 4-10 cells was also able to reduce platelet aggregation by 51.57 and 44.20% at lower concentrations (50 and 100 µM, respectively). Then, γ-TPN has good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies on the control of thromboembolic diseases.

Lysophosphatidic acid (LPA) receptor-mediated signaling regulates hypoxia-induced biological functions of lymphatic endothelial cells

The tumor microenvironment is an extremely complex composed of cancer cells and various non-cancer cells, including lymphatic endothelial cells. Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) activate a variety of malignant properties in human malignancies. In the present study, we examined the roles of LPA receptor-mediated signaling in biological responses of lymphatic endothelial SVEC4-10 cells induced by hypoxia. Lpar1, Lpar2 and Lpar3 expressions were decreased in SVEC4-10 cells cultured at hypoxic conditions (1 % O2). LPA had no impact on the cell growth activity of SVEC4-10 cells in 21 % O2 culture conditions. Conversely, the cell growth activity of SVEC4-10 cells in 1 % O2 culture conditions was reduced by LPA. The cell motile activity of SVEC4-10 cells was elevated by 1 % O2 culture conditions. GRI-977143 (LPA2 agonist) and (2S)-OMPT (LPA3 agonist) stimulated SVEC4-10 cell motility as well as AM966 (LPA1 antagonist). In tube formation assay, the tube formation of SVEC4-10 cells in 1 % O2 culture conditions was markedly increased, in comparison with 21 % O2. GRI-977143 and (2S)-OMPT elevated the tube formation of SVEC4-10 cells. Furthermore, the tube formation of SVEC4-10 cells was increased by AM966. These results suggest that LPA receptor-mediated signaling contributes to the modulation of hypoxic-induced biological functions of lymphatic endothelial cells.

Vascular endothelial dysfunction induced by 3-bromofluoranthene via MAPK-mediated-NFκB pro-inflammatory pathway and intracellular ROS generation

3-Bromofluoranthene (3-BrFlu) is the secondary metabolite of fluoranthene, which is classified as a polycyclic aromatic hydrocarbon, through bromination and exists in the fine particulate matter of air pollutants. Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular and vascular diseases. Little is known about the molecular mechanism of 3-BrFlu on endothelial dysfunction in vivo and in vitro assay. In the present study, 3-BrFlu included concentration-dependent changes in ectopic angiogenesis of the sub-intestinal vein and dilation of the dorsal aorta in zebrafish. Disruption of vascular endothelial integrity and up-regulation of vascular endothelial permeability were also induced by 3-BrFlu in a concentration-dependent manner through pro-inflammatory responses in vascular endothelial cells, namely, SVEC4-10 cells. Generation of pro-inflammatory mediator PGE2 was induced by 3-BrFlu through COX2 expression. Expression of COX2 and generation of pro-inflammatory cytokines, including TNFα and IL-6, were induced by 3-BrFlu through phosphorylation of NF-κB p65, which was mediated by phosphorylation of MAPK, including p38 MAPK, ERK and JNK. Furthermore, generation of intracellular ROS was induced by 3-BrFlu, which is associated with the down-regulated activities of the antioxidant enzyme (AOE), including SOD and catalase. We also found that 3-BrFlu up-regulated expression of the AOE and HO-1 induced by 3-BrFlu through Nrf-2 expression. However, the 3-BrFlu-induced upregulation of AOE and HO-1 expression could not be revised the responses of vascular endothelial dysfunction. In conclusion, 3-BrFlu is a hazardous substance that results in vascular endothelial dysfunction through the MAPK-mediated-NFκB pro-inflammatory pathway and intracellular ROS generation.

Rosuvastatin Enhances Lymphangiogenesis after Myocardial Infarction by Regulating the miRNAs/Vascular Endothelial Growth Factor Receptor 3 (miRNAs/VEGFR3) Pathway.

Several clinical studies have suggested that the early administration of statins could reduce the risk of in-hospital mortality in acute myocardial infarction (AMI) patients. Recently, some studies have identified that stimulating lymphangiogenesis after AMI could improve cardiac function by reducing myocardial edema and inflammation. This study aimed to identify the effect of rosuvastatin on postinfarct lymphangiogenesis and to identify the underlying mechanism of this effect. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in mice orally administered rosuvastatin for 7 days. The changes in cardiac function, pathology, and lymphangiogenesis following MI were measured by echocardiography and immunostaining. EdU, Matrigel tube formation, and scratch wound assays were used to evaluate the effect of rosuvastatin on the proliferation, tube formation, and migration of the lymphatic endothelial cell line SVEC4-10. The expression of miR-107-3p, miR-491-5p, and VEGFR3 was measured by polymerase chain reaction (PCR) and Western blotting. A gain-of-function study was performed using miR-107-3p and miR-491-5p mimics. The rosuvastatin-treated mice had a significantly improved ejection fraction and increased lymphatic plexus density 7 days after MI. Rosuvastatin also reduced myocardial edema and inflammatory response after MI. We used a VEGFR3 inhibitor to partially reverse these effects. Rosuvastatin promoted the proliferation, migration, and tube formation of SVEC4-10 cells. PCR and Western blot analyses revealed that rosuvastatin intervention downregulated miR-107-3p and miR-491-5p and promoted VEGFR3 expression. The gain-of-function study showed that miR-107-3p and miR-491-5p could inhibit the proliferation, migration, and tube formation of SVEC4-10 cells. Rosuvastatin could improve heart function by promoting lymphangiogenesis after MI by regulating the miRNAs/VEGFR3 pathway.

Sars-CoV2 ORF7a Protein Stimulates Synthesis and Secretion of Endothelial Von Willebrand Factor

Introduction: Patients with SARS-CoV-2 show significantly elevated plasma levels of von Willebrand factor (VWF) and increased thrombosis. However, the molecular mechanism underlying increased plasma levels of VWF in COVID-19 patients is not fully understood. Methods. Drosophila genetic screen, recombinant expression, endothelial culture, confocal microscopy, flow cytometry, and a microfluidic assay were employed for the study. Results. Drosophila genetic screen for SARS-CoV-2 proteins allowed us to identify Orf7a to be the key factor that induces Hml (drosophila homolog of human VWF)-dependent hypercoagulability. Recombinant Orf7a tagged with mCherry (mCh) and FLAG was expressed transiently and stably in a murine endothelial cell line SVEC4-10 and a HEK293 cell line. The ORF7a protein is localized predominantly to the Golgi and trans-Golgi network (TGN) in HEK293 cells ( Fig. 1A) but shows its expression in perinuclear area and cell surface in SVEC4-10 cells ( Fig. 1B). Significantly higher numbers of VWF-strings were formed in the ORF7a transfected SVEC4-10 cell line under static state than in the non-transfected control cells. These cell surface or extracellular VWF strings were readily removed by a human recombinant ADAMTS13 ( Fig. 1C & 1D). Flow cytometry analysis revealed that significant higher levels of intracellular VWF signal in the cells following transient expression of Orf7a than in those non-expressed controls ( Fig. 2A & 2B). Microfluidic shear-based assay and confocal imaging analysis further demonstrated that dramatically increased rate and surface coverage of fluorescent labeled murine platelets over a histone-stimulated SVEC surface expressing Orf7 comparing with over the same cells without expression of Orf7, following perfusion of Adamts13 -/- murine whole blood (anticoagulated with PPACK) under arterial shear ( Fig. 2C & 2D). Using proteomics, we identified conserved host proteins ARF4, GBF1 and ERGIC-53 (a COPII pathway component) as direct interacting factors for Orf7a and demonstrated that these proteins have conserved roles in VWF secretion. Conclusions:Our results demonstrate that ORF7a may upregulate VWF synthesis and secretion in endothelial cells, which enhances thrombus formation. Recombinant ADAMTS13 may rapidly remove endothelial surface ultra large VWF strings and exhibit therapeutic potential for COVID-19 associated thrombosis.

Vascular endothelial dysfunction induced by 3 bromofluoranthene via MAPK mediated NF B proinflammatory pathway and intracellular ROS generation

Abstract 3-Bromofluoranthene (3-BrFlu) is the secondary metabolite of fluoranthene, which is classified as a polycyclic aromatic hydrocarbon, through bromination and exists in the fine particulate matter of air pollutants. Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular and vascular diseases. Little is known about the molecular mechanism of 3-BrFlu on endothelial dysfunction in vivo and in vitro assay. In the present study, 3-BrFlu included concentration-dependent changes in ectopic angiogenesis of the sub-intestinal vein and dilation of the dorsal aorta in zebrafish. Disruption of vascular endothelial integrity and up-regulation of vascular endothelial permeability were also induced by 3-BrFlu in a concentration-dependent manner through pro-inflammatory responses in vascular endothelial cells, namely SVEC4-10 cells. Generation of pro-inflammatory mediator PGE2 was induced by 3-BrFlu through COX2 expression. Expression of COX2 and generation of pro-inflammatory cytokines, including TNFa and IL-6, were induced by 3-BrFlu through phosphorylation of NF-kB p65, which was mediated by phosphorylation of MAPK including p38 MAPK, ERK and JNK. Furthermore, generation of intracellular ROS was induced by 3-BrFlu, which is associated with the down-regulated activities of the antioxidant enzyme (AOE), including SOD and catalase. We also found that 3-BrFlu up-regulated expression of the AOE and HO-1 induced by 3-BrFlu through Nrf-2 expression. However, the 3-BrFlu-induced upregulation of AOE and HO-1 expression could not be revised the responses of vascular endothelial dysfunction. In conclusion, 3-BrFlu is a hazardous substance that results in vascular endothelial dysfunction through the MAPK-mediated-NFkB pro-inflammatory pathway and intracellular ROS generation.

Loss of ADAR1 in macrophages in combination with interferon gamma suppresses tumor growth by remodeling the tumor microenvironment

BackgroundADAR1, the major enzyme for RNA editing, has emerged as a tumor-intrinsic key determinant for cancer immunotherapy efficacy through modulating interferon-mediated innate immunity. However, the role of ADAR1 in innate...

3-Bromofluoranthene-induced cardiotoxicity of zebrafish and apoptosis in the vascular endothelial cells via intrinsic and extrinsic caspase-dependent pathways

Fluoranthene, a high-molecular-weight polycyclic aromatic hydrocarbon (PAH), is widely present in air pollutants, including fine inhalable particulate matter. 3-Bromofluoranthene (3-BrFlu), which is a brominated fluoranthene and halogenated PAH, is generated from waste combustion, metallurgical processes, cement production, e-waste dismantling, and photoreaction. Vascular endothelial cells have key functions in the homeostasis and the development of the cardiovascular system. The zebrafish model has been widely employed to study cardiotoxicity and embryotoxicity. However, no evidence has indicated that 3-BrFlu induces cytotoxicity in vascular endothelial cells, or cardiotoxicity and embryotoxicity in zebrafish. In this study, 3-BrFlu induced concentration-dependent changes in embryo- and cardiotoxicity. Cytotoxicity was also induced by 3-BrFlu in a concentration-dependent manner through apoptosis and necrosis in vascular endothelial cells, SVEC4-10 cells. The activities of caspase-3, -8, and -9 were induced by 3-BrFlu via an intrinsic pathway constituting Bcl-2 downregulation, Bad upregulation, and mitochondrial dysfunction; the extrinsic pathway included the expression of death receptors, including tumour necrosis factor α and Fas receptors. These results indicated that 3-BrFlu caused cardio- and embryotoxicity in zebrafish through vascular endothelial cells cytotoxicity resulting from caspase-dependent apoptosis through intrinsic and extrinsic pathways.

Lymphatic endothelial cells promote T lymphocyte migration into lymph nodes under hyperlipidemic conditions

Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4-10 cell line was treated with palmitic acid at a 100 μM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4-10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.

Abstract 2059: Alcohol enhances estrogen-responsive breast cancer neovascularization

Abstract The pleiotropic effects of estrogen in breast cancer development have been observed at both the initiation and progression phases. Interestingly, alcohol, as a dietary component, has been observed to possess estrogenic activity, but the synergistic interaction between alcohol and estrogen and its cellular targets remains undefined. Our previous study identified neovascularization as a cellular target of estrogen that was mediated by the mobilization of bone marrow-derived endothelial progenitor cells (BM-EPCs) and their subsequent incorporation within the tumor mass. The present study was undertaken to define the synergistic interaction between estrogen and alcohol using a cell culture model. To examine the effects of both estrogen and alcohol on breast cancer progression, we developed an in vitro model using the murine mammary tumor cell line, TG1-1, and the murine endothelial cell line, SVEC4-10. Neovascularization was assayed by the tubulogenesis and scratch wound assays as well as quantitating the expression of various provasculogenic markers. Conditioned media, generated by culturing the murine mammary cancer cell line, TG1-1, in estrogen and ethanol, was observed to enhance tubule formation and migration of the murine endothelial cell line, SVEC4-10. In addition, the TG1-1 conditioned media enhanced the prosurvival MAP kinase-signaling pathway in SVEC4-10 cells. Presumably, the observed enhancement of both endothelial cell tubulogenesis and migration is in part attributed to provasculogenic proteins secreted by TG1-1 cells. In order to address this, Western blot analysis using TG1-1 cell lysates indicated an increase in VEGF, MMP-9, and eNOS expression in presence of alcohol and estrogen. As a whole, our data suggest that both estrogen and alcohol function to enhance expression of key provasculogenic molecules in breast cancer cells, which in turn modulates the morphologic, migratory, and survival properties of endothelial cells. Citation Format: Natalie C. Cruz, Rachana Maniyar, Robert Suriano. Alcohol enhances estrogen-responsive breast cancer neovascularization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2059.

Dual-Targeted Theranostic Delivery of miRs Arrests Abdominal Aortic Aneurysm Development.

Abdominal aortic aneurysm (AAA) is an often deadly disease without medical, non-invasive treatment options. The upregulation of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium provides an early target epitope for a novel biotechnological theranostic approach. MicroRNA-126 was used as a therapeutic agent, based on its capability to downregulate VCAM-1 expression in endothelial cells and thereby reduces leukocyte adhesion and exerts anti-inflammatory effects. Ultrasound microbubbles were chosen as carriers, allowing both molecular imaging as well as targeted therapy of AAA. Microbubbles were coupled with a VCAM-1-targeted single-chain antibody (scFvmVCAM-1) and a microRNA-126 mimic (M126) constituting theranostic microbubbles (TargMB-M126). TargMB-M126 downregulates VCAM-1 expression in vitro and in an in vivo acute inflammatory murine model. Most importantly, using TargMB-M126 and ultrasound-guided burst delivery of M126, the development of AAA in an angiotensin-II-induced mouse model can be prevented. Overall, we describe a unique biotechnological theranostic approach with the potential for early diagnosis and long-sought-after medical therapy of AAA.

The Anti-Inflammatory and Vasodilating Effects of Three Selected Dietary Organic Sulfur Compounds from Allium Species.

The anti-inflammatory and vasodilating effects of three selected dietary organic sulfur compounds (OSC), including diallyl disulfide (DADS), dimethyl disulfide (DMDS), and propyl disulfide (PDS), from Allium species were investigated. In the anti-inflammatory activity assay, the three OSC demonstrated significant inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced RAW 264.7 cells. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in activated RAW 264.7 cells was inhibited by the three OSC, indicating that the three OSC prevented the LPS-induced inflammatory response in RAW 264.7 cells. For the vasodilative assay, the three OSC were ineffective in producing NO in SVEC4-10 cells, but they did enhance prostacyclin (PGI2) production. The expression of COX-2 in SVEC4-10 cells was activated by DADS and DMDS. Pretreatment of SVEC4-10 cells with the three OSC decreased ROS generation in H2O2-induced SVEC4-10 cells. In addition, the three OSC significantly inhibited angiotensin-I converting enzyme (ACE). The up-regulation of PGI2 production and COX-2 expression by DADS and DMDS and the reduction of ROS generation by DADS, DMDS, and PDS in SVEC4-10 cells contributed to the vasodilative effect of the three OSC. Collectively, these findings suggest that DADS, DMDS, and PDS are potential anti-inflammatory and vasodilative mediators.

Abstract 3365: Provasculogenic effects of alcohol and estrogen: implications for breast cancer development

Abstract Over 200,000 cases of breast cancer are diagnosed within the United States, resulting in over 40,000 deaths annually. Of the many lifestyle habits that are associated with cancer development, various epidemiological observations have provided a strong link between increased alcohol consumption and breast cancer progression. Specifically, this observation was made in women on estrogen replacement therapy thus suggesting a possible synergistic link between alcohol and estrogen. Our laboratory has demonstrated that estrogen increases breast cancer neo-vascularization both in vivo and in vitro. Provided that alcohol and estrogen can potentially synergize to promote breast cancer progression, we hypothesize that this progression is attributed to an increase in tumor neo-vascularization. In the current study, Tg-1 murine mammary carcinoma cells were treated with varying concentrations of alcohol +/- estrogen and analyzed for angiogenesis markers, such as VEGF and eNOS, and the pro-proliferation marker, MEK. At 24 hours post treatment, VEGF and eNOS were upregulated in response to both estrogen and alcohol compared to untreated cells and cells treated with either alcohol or estrogen alone. MEK levels remained constant through the conditions with a marginal upregulation in presence of alcohol and estrogen when compared to untreated cells. To further characterize neo-vascularization, we performed a scratch wound assay using the murine endothelial cell line, SVEC4-10, which was cultured in Tg1-1 conditioned media. The scratch wound assay results indicated enhanced migration of endothelial cells in response to conditioned media from Tg1-1 cells cultured in the presence of both alcohol and estrogen. SVEC4-10, treated with Tg-1-1 conditioned media were processed by Western blot analyses for MEK expression. MEK was upregulated in SVEC4-10 cells cultured in the media obtained from cells cultured with both alcohol and estrogen, indicating enhanced proliferation. Our results indicate a pro-neovasculogenic effect elicited by alcohol and estrogen, as determined by enhanced proliferation and migration of endothelial as well as increased expression of the pro-vasculogenic markers, VEGF and eNOS. Future experiments will be aimed to characterize the expression of other essential pro-vasculogenic markers, such as basic fibroblast growth factor (bFGF) and angiopoietin (Ang). The identification of these cellular and metabolic markers will establish a biochemical link between estrogen activity and signal transduction induced cellular effects. Citation Format: Rachana R. Maniyar, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Neha Tuli, Abraham Mittelman, Raj K. Tiwari, Robert Suriano. Provasculogenic effects of alcohol and estrogen: implications for breast cancer development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3365.

Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy

We recently reported that the Ins2Akita mouse is a good model for late-onset diabetic retinopathy. Here, we investigated the effect of miR200-b, a potential anti-angiogenic factor, on VEGF receptor 2 (VEGFR-2) expression and to determine the underlying angiogenic response in mouse endothelial cells, and in retinas from aged Ins2Akita mice. MiR200-b and its native flanking sequences were amplified and cloned into a pCAG-eGFP vector directed by the ubiquitous CAG promoter (namely pCAG-miR200-b-IRES-eGFP). The plasmid was compacted by CK30PEG10K into DNA nanoparticles (NPs) for in vivo delivery. Murine endothelial cell line, SVEC4-10, was first transfected with the plasmid. The mRNA levels of VEGF and VEGFR-2 were quantified by qRT-PCR and showed significant reduction in message expression compared with lipofectamine-transfected cells. Transfection of miR200-b suppressed the migration of SVEC4-10 cells. There was a significant inverse correlation between the level of expression of miR200-b and VEGFR-2. Intravitreal injection of miR200-b DNA NPs significantly reduced protein levels of VEGFR-2 as revealed by western blot and markedly suppressed angiogenesis as evaluated by fundus imaging in aged Ins2Akita mice even after 3months of post-injection. These findings suggest that NP-mediated miR200-b delivery has negatively regulated VEGFR-2 expression in vivo.

Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization.

Brain endothelial IL-1R1 mediates central IL-1 induced neuroinflammation and anxiety-like behavior

Interleukin-1 (IL-1) mediates diverse neuropathological effects and behavioral changes in the central nervous system (CNS) through type I IL-1 receptor (IL-1R1). Different CNS cell types are known to mediate different IL-1 effects. In this study, we investigated IL-1 induced neuroinflammation and anxiety using three cell-type specific IL-1R1 expressing mouse lines: Tie2Cre-IL-1R1r/r (IL-1R1 restricted to endothelial cells and hematopoietic cells), LysMCre-IL-1R1r/r (myeloid cells) or HipAAV2Cre-IL-1R1r/r (hippocampal neurons). Following intracerebroventricular (ICV) IL-1 injection microglial activation was induced in all three mouse lines but with distinct morphological characteristics. Microglia in Tie2Cre-IL-1R1r/r mice developed most abundant branches and longest processes length. Cytokine expression analysis showed microglia in wild-type (WT) and Tie2Cre-IL-1R1r/r mice upregulated proinflammatory cytokines IL-1 β and TNF- α , which did not occur in LysMCre-IL-1R1 and AAVCre-IL-1R1 mice which lacked endothelial IL-1R1. We then adoptively transferred SVEC4-10 cells, an IL-1R1 expressing endothelial cell line, into striatum of IL-1R1 knockout mice (IL-1R1r/r). After the transfer, microglial activation was induced by ICV IL-1 but not by saline, indicating endothelial IL-1R1 is sufficient to mediate inflammatory microglial activation. Furthermore, we found endothelial IL-1R1 dependent microglial activation was mediated via exocytosis of danger signals such as ATP. In addition, anxiety-like behavior developed in WT and Tie2Cre-IL-1R1r/r mice but not LysMCre-IL-1R1r/r or AAV2Cre-IL-1R1r/r lines. Collectively, these findings demonstrate that brain endothelial IL-1R1 mediates central IL-1 induced neuroinflammation and anxiety-like behavior.

Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma.

Endoglin (CD105), a transforming growth factor (TGF)-β coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.

Reduction of adhesion molecule production and alteration of eNOS and endothelin-1 mRNA expression in endothelium by Euphorbia hirta L. through its beneficial β-amyrin molecule.

The inflammatory reaction in large blood vessels involves up-regulation of vascular adhesion molecules such as endothelial cell selectin (E-selectin), soluble vascular cell adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. These vascular dysfunctions are associated with the development of atherosclerosis. β-Amyrin, an active component of Euphorbia hirta L., has potent anti-inflammatory effects. So far, its preventive effects against the expression of inflammatory mediator-induced adhesion molecules have not been investigated. Endothelial cells (SVEC4-10 cell line) were treated with 50% RAW conditioned media (i.e., normal SVEC4-10 culture media contains 50% of lipopolysaccharide-activated macrophage culture media) without or with β-amyrin (0.6 and 0.3 µM). The production levels of E-selectin, sICAM-1, and sVCAM-1 in the SVEC4-10 cells were measured with ELISA assay kits. Under the same treatment conditions, expression of endothelin (ET)-1 and endothelial type of NO synthase (eNOS) mRNA were analyzed by RT-PCR and agarose gel. With β-amyrin, the 50% RAW conditioned media-induced E-selectin, sICAM-1, and sVCAM-1 levels as well as ET-1 gene expression were all suppressed. β-Amyrin treatment also restored the 50% RAW conditioned media-suppressed eNOS mRNA expression. These data indicate that β-amyrin is potentially useful in preventing chronic inflammation-related vascular diseases.

Lymphotoxin Is Required for Treg Migration and Function for Islet Allograft Survival.

Introduction: Treg migration from islet allografts to draining LN is essential for prolonging allograft survival. Lymphotoxin (LT) regulates the organization, development, and maintenance of lymphoid organs, and LT beta receptor (LTβR) signaling has been implicated in T cell migration in the thymus. However, the role of LT in T cell migration to lymph node (LN) has not been explored. Here we tested the hypothesis that LT- LTβR interactions are important for Treg migration to LN and essential for prolonging graft survival. Methods: T cell subsets were generated using Foxp3GFP mice, and purified based on CD44, CD25, and Foxp3GFP expression, or isolated directly from wild type (WT) C57BL/6 or lymphotoxin-α deficient mice (LTα KO) using CD4 and CD25 bead selection. T cells were used in transmigration assays across lymphatic endothelial SVEC4-10 or blood endothelial MS-1 cell lines. For in vivo assays, T cells were labeled with tracking dyes, injected via the foot pad, intravenously, into the ear pinna, or co-transferred with BALB/c islet allografts under the kidney capsule, and analyzed at various times and sites. LTβR fusion protein (LTβRIg) was used to block LT interactions with LTβR. Results: WT but not LTa KO Treg prolonged islet allograft function. LTα KO Treg suppressed responder T cell proliferation normally in vitro, but failed to migrate from islet grafts to dLN. WT Treg migration from footpads to popliteal dLN was inhibited by LTβRIg, while non-Treg CD4 T cell migration was unaffected. In contrast, Treg treated with LTβRIg migrated from blood directly into LN as well as controls. Both natural and induced Treg migration to lymphatics was inhibited by LTβRIg, while non Treg CD4 T cell migration was not inhibited. Treg transmigration across lymphatic endothelial SVEC4-10 cells but not blood endothelial MS-1 cells in vitro was inhibited by LTβRIg, while transmigration of non-Treg CD4 T cells was not affected. Conclusions: Treg, but not non-Treg CD4 T cells, rely upon stimulation of LTβR on lymphatic endothelium for migration to LN via afferent lymphatics. This interaction is not required for LN entry from blood through HEV. This reveals a previously undescribed role for LTβR in regulating Treg entry into LN. Since Treg are specialized to engage afferent lymphatics to enter LN and be fully suppressive, modulating this pathway may allow targeted disruption or enhancement of Treg migration to LN and suppress or enhance immunity or tolerance.