The mouse cornea micropocket angiogenesis assay

Rogers, Michael S.; Birsner, Amy E.; D’Amato, Robert J.

doi:10.1038/nprot.2007.368

Cited by 109 publications

(102 citation statements)

References 21 publications

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“…pocket assay (30). Systemic administration of the bispecific Ang-2-VEGF CrossMab CH1-CL at a dose of 10 mg/kg was able to abolish completely VEGF-induced corneal angiogenesis from the limbus toward the VEGF gradient in a manner comparable to the combination of bevacizumab and LC06; bevacizumab alone exerted a less pronounced effect (Fig.…”

Section: Resultsmentioning

confidence: 99%

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Schaefer

Regula²,

Bähner³

et al. 2011

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

397

371

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We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

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Section: Resultsmentioning

confidence: 99%

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Schaefer

Regula²,

Bähner³

et al. 2011

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

397

371

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“…12 Neoangiogenesis in HCCLM3-derived, shRNA-CD151-HCCLM3-derived, shRNA-MMP9-HCCLM3-derived, HCCLM3-mockderived, and Hep3B-derived tumor tissues was assayed with the mouse cornea micropocket angiogenesis model as described previously. 24 The vessel area was calculated as follows: 6 HCCLM3, shRNA-CD151-HCCLM3, shRNA-MMP9-HCCLM3, HCCLM3-mock, and Hep3B cells were used for spontaneous metastasis assays, as described previously. 25 Lung metastases of shRNA-CD151-HCCLM3, shRNA-MMP9-HCCLM3, and HCCLM3-mock were visualized with fluorescence stereomicroscopy (Leica Microsystems Imaging Solutions, Ltd., Cambridge, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

CD151 Modulates Expression of Matrix Metalloproteinase 9 and Promotes Neoangiogenesis and Progression of Hepatocellular Carcinoma

et al. 2010

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Tetraspanin CD151 is involved in several pathological activities associated with tumor progression, including neoangiogenesis. However, the role and molecular mechanism of CD151 in the neoangiogenesis of hepatocellular carcinoma (HCC) remain enigmatic. We found that the level of expression of matrix metalloproteinase 9 (MMP9) was positively associated with CD151 expression in HCC cells. We developed a zone-by-zone blockade and demonstrated that overexpression of CD151 in HCC cells facilitated MMP9 expression through a phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3b (GSK-3b)/Snail signaling pathway. In contrast, down-regulation of CD151 expression impaired the ability of HCC cells to form microvessels in vitro and reduced their in vivo metastatic potential. In a clinical setting, a significant correlation of the expression of CD151 with MMP9 expression and with microvessel density (MVD) was revealed by Pearson correlation analysis of HCC patients. The postoperative 3-, 5-, and 7-year overall survival rates of HCC patients with CD151 high in comparison with the other groups. Multivariate Cox proportional hazards analysis showed that the concomitant overexpression of CD151, MMP9, and MVD was an independent marker for predicting poor prognosis of HCC. Conclusion: Overexpression of CD151 up-regulated the expression of MMP9 through the PI3K/Akt/GSK-3b/Snail pathway. CD151-dependent neoangiogenesis appeared to promote the progression of HCC, and this suggests that CD151 may be useful as a high-priority therapeutic target for antiangiogenesis in HCC. (HEPATOLOGY 2010;52:183-196) Abbreviations: AFP, alpha-fetoprotein; AKT, protein kinase B; bFGF, basic fibroblast growth factor; CDC42, cell division control protein 42 homologue; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSK, glycogen synthase kinase; H&E, hematoxylin and eosin; HBsAg, hepatitis B surface antigen; HCC, hepatocellular carcinoma; HR, hazard ratio; HUVEC, human umbilical vein endothelial cell; LY294002, 2-morpholin-4-yl-8-phenylchromen-4-one; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; mRNA, messenger RNA; MVD, microvessel density; NA, not adopted; NS, not significant; OS, overall survival; PI3K, phosphatidylinositol-3-kinase; qRT-PCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; shRNA, short hairpin RNA; siRNA, small interfering RNA; TNM, tumor node metastasis; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; VEGF, vascular endothelial growth factor.From the

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“…Vessel area was calculated using the formula VA ϭ 0.02͓͔ ϫ VL ϫ CH where VA is the vessel area (mm 2 ), VL is the vessel length, and CH is the clock hour as described previously. 18 …”

Section: Corneal Micropocket Assaymentioning

confidence: 99%

Cross-Talk between Vascular Endothelial Growth Factor and Matrix Metalloproteinases in the Induction of Neovascularization in Vivo

Ebrahem

Chaurasia

Vasanji

et al. 2010

The American Journal of Pathology

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Matrix metalloproteinases (MMPs), a specialized group of enzymes capable of proteolytically degrading extracellular matrix proteins , have been postulated to play an important role in angiogenesis. It has been suggested that MMPs can regulate neovascularization using mechanisms other than simple remodeling of the capillary basement membrane. To determine the interplay between vascular endothelial growth factor (VEGF) and MMPs , we investigated the induction of angiogenesis by recombinant active MMPs and VEGF in vivo. Using a rat corneal micropocket in vivo angiogenesis assay , we observed that the active form of MMP-9 could induce neovascularization in vivo when compared with the pro-form of the enzyme as a control. This angiogenic response could be inhibited by neutralizing VEGF antibody , which suggests that MMPs acts upstream of VEGF. Additional in vitro studies using extracellular matrix loaded with radiolabeled VEGF determined that active MMPs can enzymatically release sequestered VEGF. Interestingly, in vivo angiogenesis induced by VEGF could be inhibited by MMP inhibitors , indicating that MMPs also act downstream of VEGF. In addition , inflammation plays an important role in the induction of angiogenesis mediated by both VEGF and MMPs. Our results suggest that MMPs act both upstream and downstream of VEGF and imply that potential combination therapies of VEGF and MMP inhibitors may be a useful therapeutic approach in diseases of pathological neovascularization.

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The mouse cornea micropocket angiogenesis assay

Cited by 109 publications

References 21 publications

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

CD151 Modulates Expression of Matrix Metalloproteinase 9 and Promotes Neoangiogenesis and Progression of Hepatocellular Carcinoma

Cross-Talk between Vascular Endothelial Growth Factor and Matrix Metalloproteinases in the Induction of Neovascularization in Vivo

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