Abstract 012: Mineralocorticoid Receptors Mediate Western Diet-Induced Macrophage Polarization and Aortic Stiffness

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Guanghong Jia ◽  
Annayya R Aroor ◽  
Vincent G DeMarco ◽  
Zhe Sun ◽  
Brian P Bostick ◽  
...  
Keyword(s):  
Aortic Stiffness ◽  
Low Dose ◽  
Ex Vivo ◽  
Macrophage Polarization ◽  
Western Diet ◽  
Marker Genes ◽  
Mineralocorticoid Receptors ◽  
Expression Ratio

Mineralocorticoid receptors (MRs) are expressed in vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells (ECs) and signaling through these receptors may play a key role in development of aortic stiffness. Accordingly, we hypothesized that these vascular MRs are involved in the western diet (WD) - induced macrophage polarization and aortic stiffness. The stiffness of primary cultured VSMC and tissue ECs or VSMCs from mice was measured using cell nano-indentation with atomic force microscopy (AFM). Both Ang II (100 nM) and aldosterone (10 nM) increased VSMC stiffness in vitro. Also, B57 mice fed a WD for 16 weeks increased EC and VSMC stiffness as determined by in vivo pulse wave velocity and ex vivo AFM techniques. The elevated aortic stiffness was accompanied by increased mRNA expression of M1 markers MCP-1, CD 86, and CD11b. However, treatment with low dose spironolactone (Sp) (1.0 mg/kg/day), a dose not affecting blood pressure, significantly attenuated WD-induced increases in stiffness in murine ECs and VSMCs. Sp increased M2 marker IL10 and the expression ratio of M2/M1 marker genes in aorta by real time PCR. Interestingly, mice with EC MR knockout did not develop WD- induced aortic vasodilation dysfunction under both Ach and SNP (10-9-10-4 mol/L) stimulation. Furthermore, low dose Sp inhibited WD- caused up-regulation of ERK 1/2 and down-regulation Akt/eNOS signaling pathways in the aorta. These findings support the notion that increased vascular and macrophage MR signaling play a key role in macrophage polarization and associated aortic stiffness that result from consuming a WD high in fructose and fat.

scholarly journals Conditionally Reprogrammed Cells from Patient-Derived Xenograft to Model Neuroendocrine Prostate Cancer Development

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1398 ◽  
Author(s):  
Xinpei Ci ◽  
Jun Hao ◽  
Xin Dong ◽  
Hui Xue ◽  
Rebecca Wu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Ex Vivo ◽  
Cancer Cell Line ◽  
Marker Genes ◽  
Patient Derived Xenograft ◽  
Pdx Model ◽  
Neuroendocrine Prostate Cancer ◽  
Parental Tumor

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo.

scholarly journals microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression

2020 ◽  
Vol 11 ◽  
Author(s):  
Stephen Fitzsimons ◽  
Silvia Oggero ◽  
Robyn Bruen ◽  
Cathal McCarthy ◽  
Moritz J. Strowitzki ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Macrophage Polarization ◽  
Chronic Inflammatory Disease ◽  
Cholesterol Diet ◽  
Human Coronary Artery ◽  
Inflammatory Signaling ◽  
Anti Inflammatory

BackgroundAtherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD).MethodsmiR-155 expression was quantified in aortae from ApoE−/− mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD.ResultsHere, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs.ConclusionmiR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

scholarly journals RAGE antagonist peptide mitigates AGE-mediated endothelial hyperpermeability and accumulation of glycoxidation products in human ascending aortas and in a murine model of aortic aneurysm

2021 ◽  
Author(s):  
Chiara Camillo ◽  
Alexey Abramov ◽  
Philip M Allen ◽  
Estibaliz Castillero ◽  
Emilia Roberts ◽  
...  
Keyword(s):  
Aortic Stiffness ◽  
Aortic Wall ◽  
Ex Vivo ◽  
Endothelial Permeability ◽  
Ascending Aorta ◽  
Vascular Stiffness ◽  
Ecm Remodeling ◽  
Pressure Myograph

Background: Aortic dissection and aneurysm are the result of altered biomechanical forces associated with structural weakening of the aortic wall caused by genetic or acquired factors. Current guidelines recommend replacement of the ascending aorta when the diameter is >5.5 cm in tricuspid aortic valve patients. Aortopathies are associated with altered wall stress and stiffness as well as endothelial cell dysfunction and synthetic vascular smooth muscle cell (VSMC) phenotype. We reported that these mechanisms are mediated by glycoxidation products [Reactive oxygen species (ROS) and Advance Glycation End products (AGE)]. This study addresses the role of glycoxidation on endothelial function and AGE-mediated aortic stiffness. Hypothesis and aims: Here we investigate how circulating glycation products infiltrate the aortic wall via AGE-mediated endothelial hyperpermeability and contribute to both VSMC synthetic phenotype and extracellular matrix (ECM) remodeling in vivo and ex vivo. We also study how RAGE antagonist peptide (RAP) can rescue the effect of AGEs in vitro and in vivo in eNOS-/- vs WT mice. Methods and results: Human ascending aortas (n=30) were analyzed for AGE, ROS, and ECM markers. In vitro glycation was obtained by treating VSMC or human and murine aortas with glyoxal. Endothelial permeability was measured under glycation treatment. Vascular stiffness was measured by a pressure myograph comparing wild-type mice in the absence of presence of glyoxal. eNOS-/- mice, a model of increased endothelial permeability, were treated for 28 days with hyperlipidemic diet and Angiotensin II (1000ng/kg/min) with or without anti-glycation treatment (RAP 20mg/kg). Echo data of aortic diameter were collected. Murine vascular stiffness was measured by a pressure myograph (n=5/group). Glycoxidation products were detected in all human aortas independently of aortic diameter, with stronger accumulation on the lumen and the adventitia layer. AGEs increased endothelial permeability, induce synthetic phenotypic switch in human VSMCs, and inhibit cell migration. RAP pre-treatment rescue the effect of glyoxal on endothelial cells. Ex vivo glycation treatment of murine arteries impacted on ECM and increased stiffness. Aortic stiffness was higher in eNOS-/- vs WT mice. Ang II-mediated aortopathies results in aortic dilation, and AGE/ROS accumulation, which is rescued by RAGE antagonist peptide treatment of eNOS-/- mice. Conclusions: Glycoxidation reaction mediates EC permeability, VSMCs phenotype, and ECM remodeling leading to dysfunctional microstructure of the ascending aorta, altered vascular stiffness and increasing aortic susceptibility to dilation and rupture. Moreover, we show that RAP can mitigate AGE-mediated endothelial hyper-permeability in vitro and impact on ascending aneurysm in vivo

Transcriptomal comparison of human dermal lymphatic endothelial cells ex vivo and in vitro

2007 ◽  
Vol 28 (2) ◽  
pp. 179-192 ◽  
Author(s):  
Nikolaus Wick ◽  
Pipsa Saharinen ◽  
Juha Saharinen ◽  
Elisabeth Gurnhofer ◽  
Carl W. Steiner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular System ◽  
Vascular Endothelial Cells ◽  
Ex Vivo ◽  
Lymphatic Endothelial Cell ◽  
Marker Genes ◽  
Lymphatic Endothelial Cells ◽  
Extracellular Matrix Components

The in vivo functions of lymphatic endothelial cells depend on their microenvironment, which cannot be fully reproduced in vitro. Because of technical limitations, gene expression in uncultured, “ex vivo” lymphatic endothelial cells has not been characterized at the molecular level. We combined tissue micropreparation and direct cell isolation with DNA chip experiments to identify 159 genes differentiating human lymphatic endothelial cells from blood vascular endothelial cells ex vivo. The same analysis performed with cultured primary cells revealed that only 19 genes characteristic for lymphatic endothelium ex vivo retained this property upon culture, while 27 marker genes were newly induced. In addition, a set of panendothelial genes could be recognized. The propagation of lymphatic endothelial cells in culture stimulated transcription of genes associated with cell turnover, basic metabolism, and the cytoskeleton. On the other hand, there was downregulation of genes encoding extracellular matrix components, signaling via transmembrane tyrosine kinase pathways and the chemokine (C-C) ligand 21. Direct ex vivo analysis of the lymphatic endothelial cell transcriptome is helpful for the understanding of the physiology of the lymphatic vascular system and of the pathogenesis of its diseases.

scholarly journals OR17-06 Transglutaminase 2 Inhibition Reduces Aortic Stiffness in Western Diet-Fed Female Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Bhavana Chinnakotla ◽  
Camila Margarita Manrique Acevedo ◽  
Padilla Jaume ◽  
Makenzie L Woodford ◽  
Annayya R Aroor ◽  
...  
Keyword(s):  
Aortic Stiffness ◽  
Weight Control ◽  
Ex Vivo ◽  
Transglutaminase 2 ◽  
Competitive Inhibitor ◽  
Tolerance Test ◽  
Western Diet ◽  
Arterial Stiffening ◽  
Female Mice

Abstract Widespread consumption of diets high in fat, sugars and salt (Western diet, WD) is associated arterial stiffening, which is a major independent risk factor for cardiovascular disease (CVD). Notably, while WD feeding increases the risk of CVD in both males and females, the latter are more prone to develop arterial stiffening. However, the mechanisms underlying WD-induced arterial stiffening are poorly understood, particularly in females, and there are currently no specific treatments targeted at vascular stiffening.Tissue transglutaminase 2 (TG2) is an enzyme that mediates the cross-linking and stabilization of extracellular matrix proteins such as collagen, and promotes the polymerization of actin stress fibers of the cytoskeleton. It is ubiquitously expressed and abundantly present in the vasculature. Mounting evidence implicates TG2 activation in the pathogenesis of arterial stiffening and vascular fibrosis. Herein we propose that TG2 activation is central to WD-induced arterial stiffening and sought to determine the efficacy of cystamine (a non-specific competitive inhibitor of TG2) for reducing arterial stiffening in the setting of WD consumption. Accordingly, we fed 20 female mice (4 weeks old) a WD (4.65 kcal/g of food, fat 46% kcals, high-fructose corn syrup 17.5%, sucrose 17.5%, protein 17.6%, salt 1.6%) for 43 weeks. Ten of these mice received cystamine (40 mg/Kg/d in the drinking water) during their last 8 weeks on the WD. Another group of female mice (n=10) fed regular chow was used as reference controls. Aortic stiffness was measured in vivo via ultrasound-based pulse wave velocity and ex vivo by aortic explant atomic force microscopy. Vasomotor responses were assessed in isolated aortic rings via wire myography.Cystamine did not influence glucose homeostasis (intraperitoneal glucose tolerance test) or blood pressure (tail-cuff) (control 77.208±2.229 mm Hg versus WD 77.208±6.077 versus WD+Cystamine 76.297±7.894), but it was associated with increased body weight (control 26.860±2.215 grams versus WD 25.320±2.889 versus WD+Cystamine 33.220±4.848, p<0.05). Notably, cystamine reduced aortic stiffness in WD-fed mice both in vivo and ex vivo such that differences between chow-fed and WD-fed mice were normalized (control 5.294±1.713 versus WD 11.735±5.962 p≤0.05, control 5.294±1.713 versus WD+Cystamine 3.940±0.378 KPa, p<0.05). In addition, WD-induced impairments in endothelium-independent vasorelaxation (i.e. responses to sodium nitroprusside) were restored with cystamine. Collectively, our data show that cystamine reduces aortic stiffness and improves endothelium-independent vasorelaxation in female mice chronically exposed to WD, and that these effects occur despite an increase in weight gain. These findings implicate TG2 as a promising therapeutic target for reducing arterial stiffening in the context of chronic over-nutrition in females.

scholarly journals Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Zineb Belcaid ◽  
Cor Berrevoets ◽  
John Choi ◽  
Edward van Beelen ◽  
Eftychia Stavrakaki ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cell Cultures ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Ex Vivo ◽  
Single Agent ◽  
Immune Checkpoints

Abstract Background The tumor-selective human adenovirus Delta24-RGD is currently under investigation in phase II clinical trials for patients with recurrent glioblastoma (GBM). To improve treatments for patients with GBM, we explored the potential of combining Delta24-RGD with antibodies targeting immune checkpoints. Methods C57BL/6 mice were intracranially injected with GL261 cells and treated with a low dose of Delta24-RGD virus. The expression dynamics of 10 co-signaling molecules known to affect immune activity was assessed in tumor-infiltrating immune cells by flow cytometry after viral injection. The antitumor activity was measured by tumor cell killing and IFNγ production in co-cultures. Efficacy of the combination viro-immunotherapy was tested in vitro and in the GL261 and CT2A orthotopic mouse GBM models. Patient-derived GBM cell cultures were treated with Delta24-RGD to assess changes in PD-L1 expression induced by virus infection. Results Delta24-RGD therapy increased intratumoral CD8+ T cells expressing Inducible T-cell co-stimulator (ICOS) and PD-1. Functionality assays confirmed a significant positive correlation between tumor cell lysis and IFNγ production in ex vivo cultures (Spearman r = 0.9524; P < .01). Co-cultures significantly increased IFNγ production upon treatment with PD-1 blocking antibodies. In vivo, combination therapy with low-dose Delta24-RGD and anti-PD-1 antibodies significantly improved outcome compared to single-agent therapy in both syngeneic mouse glioma models and increased PD-1+ tumor-infiltrating CD8+ T cells. Delta24-RGD infection induced tumor-specific changes in PD-L1 expression in primary GBM cell cultures. Conclusions This study demonstrates the potential of using low-dose Delta24-RGD therapy to sensitize glioma for combination with anti-PD-1 antibody therapy.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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