Techniques and assays for the study of angiogenesis

Irvin, Michael; Zijlstra, Andries; Wikswo, John P.; Pozzi, Ambra

doi:10.1177/1535370214529386

Cited by 63 publications

(53 citation statements)

References 131 publications

(281 reference statements)

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“…This short‐term, reproducible assay is commonly used to test how different soluble factors affect tubulogenesis. Limitations of this assay are similar to those in other in vitro models: only certain steps of tissue function are simulated; therefore, the response may not completely reflect how angiogenesis occurs in vivo . HUVECs form tubes when stimulated by VEGF.…”

Section: Discussionmentioning

confidence: 99%

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Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro

Olekson

Rose

Carlsson

et al. 2017

Wound Repair Regeneration

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Skin quality outcome after skin grafting is adversely affected by wound bed inflammation. Neomycin, gentamicin, and other aminoglycoside antibiotics are known to modulate inflammation, and topical application affords the use of higher doses than are possible to use systemically. Previous data suggest that clinically relevant doses of neomycin, but not gentamicin, may impair angiogenesis, which is critical to the durable survival of skin grafts. The role of gentamicin at ultrahigh doses compared with clinically relevant neomycin doses in regulating inflammatory expression and angiogenesis has been examined. In a porcine skin replacement excisional wound model, continuous exposure to gentamicin increased anti-angiogenic and inflammatory expression at 7 days postgrafting. In in vitro studies, gentamicin also impaired angiogenesis in a human umbilical vein endothelial cell (HUVEC) tube formation model, increased the expression of the anti-angiogenic gene C-X-C motif chemokine 10 (CXCL10) in HUVECs and macrophages, and increased pro-inflammatory cytokine expression of macrophages in a dose-dependent manner. Neomycin exerted similar effects in vitro at clinically relevant doses on HUVEC tube formation and macrophage pro-inflammatory expression. CXCL10 was upregulated in macrophages, but did not exhibit a change in HUVECs with neomycin treatment. Ultrahigh doses of gentamicin and clinically relevant doses of neomycin affect inflammation and angiogenesis in in vivo and in vitro models. These findings suggest that topical administration of aminoglycosides have the potential to adversely influence early skin graft survival.

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Section: Discussionmentioning

confidence: 99%

“…Limitations of this assay are similar to those in other in vitro models: only certain steps of tissue function are simulated; therefore, the response may not completely reflect how angiogenesis occurs in vivo. 33 HUVECs form tubes when stimulated by VEGF. However, increasing doses of gentamicin significantly disrupt the tube-like network.…”

Section: Discussionmentioning

confidence: 99%

Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro

Olekson

Rose

Carlsson

et al. 2017

Wound Repair Regeneration

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“…Tube formation is one of the preferred methods for testing an angiogenesis response to a single factor because it is short‐term and reproducible. Because this assay is 2‐D, it does not entirely recapitulate in vivo architecture . We showed that CSA‐13‐induced tube formation is inhibited by the VEGFR2 inhibitor ZM 323881, suggesting that CSA‐13 may signal through this receptor.…”

Section: Discussionmentioning

confidence: 95%

Antimicrobial ceragenins inhibit biofilms and affect mammalian cell viability and migration in vitro

et al. 2017

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The healing of burn wounds is often hampered by bacterial infection and the formation of biofilms. Antimicrobial peptides (AMPs) are effective in promoting wound healing, but are susceptible to degradation. We have tested the ability of ceragenins (CSAs), mimics of antimicrobial peptides, to mitigate preformed biofilms and stimulate wound healing in vitro . Potent CSAs (MICs < 10 μg·mL −1 ) were tested against biofilms formed from a mixture of Pseudomonas aeruginosa and Staphylococcus aureus grown for 22 h and subjected to 20 h treatment. Many CSAs showed more potent anti‐biofilm activity than the endogenous AMP LL‐37, and CSA‐13 and CSA‐90 decreased the amount of biofilm matrix substances detected by SYPRO Ruby stain. Effects on mammalian cells were measured by viability, migration, and tube formation assays in vitro . Although CSAs were toxic to immortalized human keratinocytes (HaCaTs) at higher concentrations (>10 μg·mL −1 ), lower concentrations of CSA‐13 and CSA‐192 stimulated cell migration. CSA‐13, CSA‐90, and CSA‐142 also stimulated tube formation in an in vitro angiogenesis model. An inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) blocked tube formation stimulated by CSA‐13, suggesting that CSA‐13 signals through this receptor. Ceragenins display anti‐biofilm activity and stimulate migration and tube formation in vitro . This work suggests that ceragenins have the potential to be both topical antimicrobials and wound‐healing adjunct therapeutics.

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“…Angiogenesis is a complex process where many factors influence endothelial cells to sprout and extend . In vitro experiments are sufficient to evaluate single angiogenic factors; however, in vivo experiments should be performed with the peptides to take into account the effect of various cell types such as pericytes, extracellular matrix component, flow, tension, and pressure in the vessel …”

Section: Discussionmentioning

confidence: 99%

“…41 In vitro experiments are sufficient to evaluate single angiogenic factors; however, in vivo experiments should be performed with the peptides to take into account the effect of various cell types such as pericytes, extracellular matrix component, flow, tension, and pressure in the vessel. 42 Insulin, NT, and SP all affected crucial aspects contributing to wound healing despite not being antimicrobial under these conditions. We show that the 2 different peptides NT and insulin decrease TNFα…”

Section: Angiogenesismentioning

confidence: 99%

Neurotensin, substance P, and insulin enhance cell migration

Mouritzen

Abourayale

Ejaz

et al. 2018

Journal of Peptide Science

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Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides.

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Techniques and assays for the study of angiogenesis

Cited by 63 publications

References 131 publications

Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro

Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro

Antimicrobial ceragenins inhibit biofilms and affect mammalian cell viability and migration in vitro

Neurotensin, substance P, and insulin enhance cell migration

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