Endostatin Inhibits Microvessel Formation in the ex Vivo Rat Aortic Ring Angiogenesis Assay

Angiogenesis, the formation of new blood vessels, underlies tissue development and repair. Some medicinal plant-derived compounds can modulate the angiogenic response. Heliopsis longipes, a Mexican medicinal plant, is widely used because of its effects on pain and inflammation. The main bioactive phytochemicals from H. longipes roots are alkamides, where affinin is the most abundant. Scientific studies show various medical effects of organic extracts of H. longipes roots and affinin that share some molecular pathways with the angiogenesis process, with the vasodilation mechanism of action being the most recent. This study investigates whether pure affinin and the ethanolic extract from Heliopsis longipes roots (HLEE) promote angiogenesis. Using the aortic ring rat assay (ex vivo method) and the direct in vivo angiogenesis assay, where angioreactors were implanted in CD1 female mice, showed that affinin and the HLEE increased vascular growth in a dose-dependent manner in both bioassays. This is the first study showing the proangiogenic effect of H. longipes. Further studies should focus on the mechanism of action and its possible therapeutic use in diseases characterized by insufficient angiogenesis.

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Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530321666201230113018 ◽

2020 ◽

Vol 21 ◽

Author(s):

Hana M. Hammad ◽

Amer Imraish ◽

Maysa Al-Hussaini ◽

Malek Zihlif ◽

Amani A. Harb ◽

...

Keyword(s):

Wound Healing ◽

Rural Communities ◽

Ex Vivo ◽

Ethanol Extract ◽

Aortic Ring ◽

Treated Group ◽

Control Group ◽

Dependent Manner ◽

Achillea Fragrantissima

Objective: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. Methods: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using rat aortic ring assay and in vivo using rat excision wound model. Results: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulates the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and was found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline-treated group. This effect was comparable to that induced by MEBO, the positive control. Conclusion: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

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Rat Aortic Ring Model to Assay Angiogenesis ex vivo

BIO-PROTOCOL ◽

10.21769/bioprotoc.1622 ◽

2015 ◽

Vol 5 (20) ◽

Author(s):

Isabelle Ernens ◽

B�n�dicte Lenoir ◽

Yvan Devaux ◽

Daniel Wagner

Keyword(s):

Ex Vivo ◽

Aortic Ring ◽

Ring Model ◽

Rat Aortic Ring Model

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Preclinical Evaluation of Discorhabdins in Antiangiogenic and Antitumor Models

Marine Drugs ◽

10.3390/md16070241 ◽

2018 ◽

Vol 16 (7) ◽

pp. 241 ◽

Cited By ~ 9

Author(s):

Emily Harris ◽

Jonathan Strope ◽

Shaunna Beedie ◽

Phoebe Huang ◽

Andrew Goey ◽

...

Keyword(s):

Ex Vivo ◽

Hypoxia Inducible Factor ◽

Xenograft Model ◽

Aortic Ring ◽

Therapeutic Benefit ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

System A ◽

Oncogenic Transcription Factor

Elements of the hypoxia inducible factor (HIF) transcriptional system, a key regulator of the cellular hypoxic response, are up-regulated in a range of cancer cells. HIF is fundamentally involved in tumor angiogenesis, invasion, and energy metabolism. Inhibition of the transcriptional activity of HIF may be of therapeutic benefit to cancer patients. We recently described the identification of two marine pyrroloiminoquinone alkaloids with potent activity in inhibiting the interaction between the oncogenic transcription factor HIF-1α and the coactivator protein p300. Herein, we present further characterization data for these two screening hits: discorhabdin H (1) and discorhabdin L (2), with a specific focus on their anti-angiogenic and anti-tumor effects. We demonstrated that only discorhabdin L (2) possesses excellent anti-angiogenic activity in inhibiting endothelial cell proliferation and tube formation, as well as decreasing microvessel outgrowth in the ex vivo rat aortic ring assay. We further showed that discorhabdin L (2) significantly inhibits in vivo prostate tumor growth in a LNCaP xenograft model. In conclusion, our findings suggest that discorhabdin L (2) represents a promising HIF-1α inhibitor worthy of further drug development.

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Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

AJP Cell Physiology ◽

10.1152/ajpcell.90633.2007 ◽

2008 ◽

Vol 294 (6) ◽

pp. C1521-C1530 ◽

Cited By ~ 19

Author(s):

Shuji Kondo ◽

Yixin Tang ◽

Elizabeth A. Scheef ◽

Nader Sheibani ◽

Christine M. Sorenson

Keyword(s):

Cell Migration ◽

Vascular System ◽

Ex Vivo ◽

Vascular Development ◽

Critical Role ◽

Endothelial Cell Migration ◽

Cellular Functions ◽

Capillary Morphogenesis ◽

Angiogenesis Assay ◽

Ischemic Retinopathy

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2−/−) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2−/− mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2−/− retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2−/− mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2−/− retinal EC. Mechanistically, bcl-2−/− cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2−/− mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

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Neuropilin-1 Mediates Collapsin-1/Semaphorin III Inhibition of Endothelial Cell Motility

The Journal of Cell Biology ◽

10.1083/jcb.146.1.233 ◽

1999 ◽

Vol 146 (1) ◽

pp. 233-242 ◽

Cited By ~ 225

Author(s):

Hua-Quan Miao ◽

Shay Soker ◽

Leonard Feiner ◽

José Luis Alonso ◽

Jonathan A. Raper ◽

...

Keyword(s):

Binding Sites ◽

Aortic Ring ◽

Dependent Manner ◽

Vascular Endothelial ◽

Binding Assays ◽

Angiogenesis Assay ◽

Neuropilin 1 ◽

Endothelial Growth Factor ◽

Neuronal Guidance

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65–75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

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Abstract 358: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVCs) Maintain High Expression Levels of Angiogenic Factors and Have Superior Pro-angiogenic Effects on Endothelial Networks When Compared to Term HUCPVCs and BMSCs in an ex-vivo Rat Aortic Ring Assay

Circulation Research ◽

10.1161/res.119.suppl_1.358 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Farwah Iqbal ◽

Peter Szaraz ◽

Shlomit Kenigsberg ◽

Andree Gauthier-Fisher ◽

Clifford Librach

Keyword(s):

Ex Vivo ◽

Human Growth ◽

Aortic Ring ◽

Angiogenic Factors ◽

High Expression ◽

Loop Formation ◽

Expression Levels ◽

Vascular Regeneration ◽

Aortic Ring Assay ◽

Qpcr Array

Introduction: Features of an ideal cell type, that would be conducive to vascular regeneration include (1) expression of pro-angiogenic genes (2) secretion of factors that promote developing or regenerating vasculature, and (3) maintenance of pro-angiogenic properties in the microvascular niche. Hypothesis: FTM HUCPVCs, a young rich source of mesenchymal stromal-like cells (MSCs) are ideal candidates for vascular regeneration due to their high expression of pro-angiogenic factors. Methods: The paracrine angiogenic potential of 3 types of MSCs was evaluated and compared using ex vivo tissue culture of rat aortic rings. Aorta sections were embedded into Matrigel™, cells were added to transwell membranes (pore=0.1μm, EBM 2% FBS) and combined with aortic rings (Day 0). Radial network growth and total loop formation were monitored by microscopy. Endothelial networks were quantified by ImageJ TM software. p values were calculated using ANOVA. (N=3 experiments, n=3 replicates). At day 7, the MSCs were isolated from transwells, human cytokine gene expression levels were measured using human growth factor profiler qPCR array (Qiagen, normalizers: GAPDH, βACT). Ct>35 considered negligible. Results: In the transwell aortic ring assay, FTM HUCPVCs induced significantly greater network growths when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated rings (p≤0.05). Quantification of network loop formation showed that FTM HUCPVCs induced greater numbers of closed loops when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated networks (p≤0.0001). Human growth factor qPCR array showed a high expression of angiogenic factors (Ct<25 cycle) both at day0 and day7 of co-culture, including BMP1, GDNF, MDK, NRG1, PDGFc, VEGFa, and VEGFc. Most cytokine expression levels were maintained up to 7 days in co-culture with 1 gene upregulated (BMP6 ΔCt>3), and 3 downregulated genes (FGF19, FGF9, NRTN ΔCt>3) at day 7 when compared to day 1. Conclusion: Compared to older sources of MSCs, FTM HUCPVCs promote developing endothelial networks in vitro via paracrine mechanisms and maintain the high expression of pro-angiogenic factors when cultures with endothelial cells.

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Preparation and Analysis of Aortic Ring Cultures for the Study of Angiogenesis Ex Vivo

The Textbook of Angiogenesis and Lymphangiogenesis: Methods and Applications ◽

10.1007/978-94-007-4581-0_7 ◽

2012 ◽

pp. 127-148 ◽

Cited By ~ 4

Author(s):

Roberto F. Nicosia ◽

Giovanni Ligresti ◽

Alfred C. Aplin

Keyword(s):

Ex Vivo ◽

Aortic Ring

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Exosomes derived from induced vascular progenitor cells promote angiogenesis in vitro and in an in vivo rat hindlimb ischemia model

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00247.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. H765-H776 ◽

Cited By ~ 7

Author(s):

Takerra K. Johnson ◽

Lina Zhao ◽

Dihan Zhu ◽

Yang Wang ◽

Yan Xiao ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Ex Vivo ◽

Aortic Ring ◽

Size Exclusion ◽

Hindlimb Ischemia ◽

Vascular Progenitor Cells ◽

Angiogenic Effect

Induced vascular progenitor cells (iVPCs) were created as an ideal cell type for regenerative medicine and have been reported to positively promote collateral blood flow and improve cardiac function in a rat model of myocardial ischemia. Exosomes have emerged as a novel biomedicine that mimics the function of the donor cells. We investigated the angiogenic activity of exosomes from iPVCs (iVPC-Exo) as a cell-free therapeutic approach for ischemia. Exosomes from iVPCs and rat aortic endothelial cells (RAECs) were isolated using a combination of ultrafiltration and size-exclusion chromatography. Nanoparticle tracking analysis revealed that exosome isolates fell within the exosomal diameter (<150 nm). These exosomes contained known markers Alix and TSG101, and their morphology was validated using transmission electron microscopy. When compared with RAECs, iVPCs significantly increased the secretion of exosomes. Cardiac microvascular endothelial cells and aortic ring explants were pretreated with RAEC-Exo or iVPC-Exo, and basal medium was used as a control. iVPC-Exo exerted an in vitro angiogenic effect on the proliferation, tube formation, and migration of endothelial cells and stimulated microvessel sprouting in an ex vivo aortic ring assay. Additionally, iVPC-Exo increased blood perfusion in a hindlimb ischemia model. Proangiogenic proteins (pentraxin-3 and insulin-like growth factor-binding protein-3) and microRNAs (-143-3p, -291b, and -20b-5p) were found to be enriched in iVPC-Exo, which may mediate iVPC-Exo induced vascular growth. Our findings demonstrate that treatment with iVPC-Exo promotes angiogenesis in vitro, ex vivo, and in vivo. Collectively, these findings indicate a novel cell-free approach for therapeutic angiogenesis. NEW & NOTEWORTHY The results of this work demonstrate exosomes as a novel physiological mechanism by which induced vascular progenitor cells exert their angiogenic effect. Moreover, angiogenic cargo of proteins and microRNAs may define the biological contributors in activating endothelial cells to form a new capillary plexus for ischemic vascular diseases. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/angiogenic-exosomes-from-vascular-progenitor-cells/ .

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Contribution of Adventitia-Derived Stem and Progenitor Cells to New Vessel Formation in Tumors

Cells ◽

10.3390/cells10071719 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1719

Author(s):

Berin Upcin ◽

Erik Henke ◽

Florian Kleefeldt ◽

Helene Hoffmann ◽

Andreas Rosenwald ◽

...

Keyword(s):

Progenitor Cells ◽

Network Formation ◽

Ex Vivo ◽

Aortic Ring ◽

Angiogenesis Inhibitors ◽

Collagen Gels ◽

Tumor Vascularization ◽

Ex Vivo Model ◽

Vessel Formation ◽

Stem And Progenitor Cells

Blocking tumor vascularization has not yet come to fruition to the extent it was hoped for, as angiogenesis inhibitors have shown only partial success in the clinic. We hypothesized that under-appreciated vascular wall-resident stem and progenitor cells (VW-SPCs) might be involved in tumor vascularization and influence effectiveness of anti-angiogenic therapy. Indeed, in patient samples, we observed that vascular adventitia-resident CD34+ VW-SPCs are recruited to tumors in situ from co-opted vessels. To elucidate this in detail, we established an ex vivo model using concomitant embedding of multi-cellular tumor spheroids (MCTS) and mouse aortic rings (ARs) into collagen gels, similar to the so-called aortic ring assay (ARA). Moreover, ARA was modified by removing the ARs’ adventitia that harbors VW-SPCs. Thus, this model enabled distinguishing the contribution of VW-SPCs from that of mature endothelial cells (ECs) to new vessel formation. Our results show that the formation of capillary-like sprouts is considerably delayed, and their number and network formation were significantly reduced by removing the adventitia. Substituting iPSC-derived neural spheroids for MCTS resulted in distinct sprouting patterns that were also strongly influenced by the presence or absence of VW-SPCs, also underlying the involvement of these cells in non-pathological vascularization. Our data suggest that more comprehensive approaches are needed in order to block all of the mechanisms contributing to tumor vascularization.

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