The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

Birsner, Amy E.; Benny, Ofra; D’Amato, Robert J.

doi:10.3791/51375

Cited by 24 publications

(31 citation statements)

References 16 publications

(13 reference statements)

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“…between the new node and the original sprout location and the new node is marked as 141 'Migrating'. for the vascular front to reach the pellet is on the order of 4 days, which is consistent 145 with experimental observations [1]. A persistent, off-lattice random walk is used to 146 describe the migration of tips through the extracellular matrix of the stroma [6,7,14].…”

supporting

confidence: 65%

“…After sprouting, migrating tips, illustrated in Fig 3, are assumed to move at constant speed 91 s = 10 µm h −1 . This speed is chosen so that the average time for the vascular front to reach the 92 pellet is on the order of 4 days, which is consistent with experimental observations [1]. A 93 persistent, off-lattice random walk is used to describe the migration of tips through the 94 extracellular matrix of the stroma [6,7,14].…”

mentioning

confidence: 98%

“…The pellet is not usually located 10 on the pole of the cornea; it is shifted slightly toward the base or limbus. It is evident 11 from experimental images that the geometrical configuration of the cornea-pellet system 12 influences neovascularization patterns [1]. As shown schematically in Fig 1C, new vessel 13 formation is often focused in the region where the distance between the pellet and 14 limbus is smallest, with vessels tending to grow toward the pellet.…”

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confidence: 99%

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The importance of geometry in the corneal micropocket angiogenesis assay

Grogan

Connor

Pitt-Francis

et al. 2017

Preprint

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The corneal micropocket assay is an experimental protocol for studying vessel network formation, or neovascularization, in vivo. The assay is attractive due to the ease with which the developing vessel network can be observed in the same animal over time. However, it is difficult to relate observations of neovascularization in the hemispherical geometry of the cornea to predictions from mathematical (in silico) models and observations from in vitro models, which mostly adopt planar geometries. Here, we develop a three-dimensional, off-lattice mathematical model of neovascularization in the cornea, with a new repulsion model for the interaction of vessels with tissue boundaries. The model allows us to: i) investigate how the hemispherical geometry of the cornea influences vessel network formation and ii) identify the metrics of neovascularization that are most robust to geometrical differences between typical in silico, in vitro and in vivo tissue domains. We predict that two-dimensional geometries lead to increased vessel 'merging', or anastomosis, relative to three-dimensional versions, with differences being most pronounced when circular geometries are adopted. Cuboidal geometries lead to patterns of neovascularization that most closely mimic the in vivo case. The 'distance of the vascular front to the limbus' is found to be a robust metric of neovascularization across the studied tissue domains, while densities of tip cells and vessels and 'vascularized fraction' are more sensitive to domain choice. Given the widespread adoption and attractive simplicity of planar tissue domains, the differences identified in the present study should prove useful in relating the results of previous and future in silico and in vitro studies of neovascularization to in vivo observations in the cornea. Author summaryNeovascularization, or the formation of new blood vessels, is an important process in development, wound healing and cancer. The corneal micropocket assay is used to better understand the process and, in the case of cancer, how it can be controlled with drug therapies for improved patient outcomes. In the assay, the hemispherical shape of the cornea can influence the way the vessel network forms. This makes it difficult to directly compare results from experiments with the predictions of mathematical models or cell culture experiments, which are

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supporting

confidence: 65%

mentioning

confidence: 98%

mentioning

confidence: 99%

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The importance of geometry in the corneal micropocket angiogenesis assay

Grogan

Connor

Pitt-Francis

et al. 2017

Preprint

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“…The corneal micropocket assay was performed as previously described [20]. Briefly, a slow-release pellet containing 20ng of bFGF (R&D Systems, Minneapolis, MN, USA) was implanted in the mouse cornea ~1.0 mm from the limbus.…”

Section: Methodsmentioning

confidence: 99%

Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice

Khajavi¹,

Zhou²,

Birsner³

et al. 2017

PLoS Genet

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Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.

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“…The mouse corneal micropocket assay was performed in C57BL/6 mice as previously described. 29 Test agents included recombinant TGFBIp (40,80,160, 320 ng per pellet; R&D Systems) and VEGF-C (160 ng per pellet; PeproTech). The mouse corneal micropocket assay was surgically created on a single eye of each animal (five mice per group).…”

Section: Mouse Corneal Micropocket Assay and Pharmaceutical Intervementioning

confidence: 99%

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

Lin

Zhang

Yang

et al. 2019

J Cellular Molecular Medi

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Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor‐β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up‐regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up‐regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor‐C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1‐FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp‐integrin pathways plays a pivotal role in lymphatic sprouting.

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The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

Cited by 24 publications

References 16 publications

The importance of geometry in the corneal micropocket angiogenesis assay

The importance of geometry in the corneal micropocket angiogenesis assay

Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

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