Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices

Bayless, Kayla J.; Kwak, Hyo‐Bum; Su, Shih‐Chi

doi:10.1038/nprot.2009.221

Cited by 96 publications

(123 citation statements)

References 45 publications

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“…Our results demonstrate that the 3D sprouting of LECs into the collagen hydrogel (Movie S1) is morphologically similar to in vivo vessel sprouting as recently depicted in the mouse retina (25). We used a combination of VEGF-A, bFGF and S1P to promote lymphatic sprout formation since these factors are involved in pathological tumor and lymph node lymphangiogenesis (26,27), were previously validated in vitro (28), and yielded a more pronounced sprout formation in our assay than the individual growth factors. An advantage of our 3D assay is the automated image acquisition and analysis, which resolves these bottlenecks of conventional high-throughput screens.…”

Section: Discussionsupporting

confidence: 77%

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Schulz

Reisen

Zgraggen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds ðLOPACÞ 1280 collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.high-content screening | small compound screening | lymphatic endothelium | chemical genetics

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Section: Discussionsupporting

confidence: 77%

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Schulz

Reisen

Zgraggen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza BioProducts (San Diego, CA), maintained as described previously (49), and used at passage 4 -6.…”

Section: Methodsmentioning

confidence: 99%

Fluid Shear Stress and Sphingosine 1-Phosphate Activate Calpain to Promote Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Membrane Translocation and Endothelial Invasion into Three-dimensional Collagen Matrices

Kang

Kwak

Kaunas

et al. 2011

Journal of Biological Chemistry

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Background: Wall shear stress (WSS) and sphingosine 1-phosphate (S1P) combine to promote endothelial sprouting and angiogenesis. Results: WSS and S1P activate calpains, and calpains are required for endothelial sprouting. Blocking calpains reduced membrane type-1 matrix metalloproteinase (MT1-MMP) membrane localization. Conclusion: Calpains regulate MT1-MMP membrane localization. Significance: These data uncover a new mechanism for controlling a key metalloproteinase in angiogenesis.

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“…Primary mouse dermal endothelial cells (Celprogen Inc.) were maintained initially as recommended by the manufacturer. After the second passage, cells were passaged to flasks precoated with 1 mg/ml gelatin and fed with culture medium prepared as described previously (52). In all endothelial cells, the presence of endothelial cell markers von Willebrand factor and VEGFR was confirmed via immunohistochemistry and Western blot analysis.…”

Section: Cell Culture Human Brain Microvascular Endothelial Cells (Lmentioning

confidence: 99%

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents

Lee¹,

Clarke²,

Ahmad³

et al. 2011

J. Clin. Invest.

135

213

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Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5β1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.

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Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices

Cited by 96 publications

References 45 publications

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Fluid Shear Stress and Sphingosine 1-Phosphate Activate Calpain to Promote Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Membrane Translocation and Endothelial Invasion into Three-dimensional Collagen Matrices

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents

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