Tube formation: an in vitro matrigel angiogenesis assay.

Neovascularization plays a role in several pathological conditions, including tumor growth, arthritis, and choroidal neovascularization. Investigators from different fields can choose from several available angiogenesis assays according to their specific needs. This chapter describes an easy-to-perform assay that is based on the differentiation of endothelial cells and the formation of tube-like structures on an extracellular matrix, Matrigel. The assay can be used to screen compounds for angiogenic activity or to determine if it has an effect on angiogenesis, depending on the conditions chosen. It is a quick assay, easy to set up, and highly reproducible. It can be used to test one or two samples, or it can quickly be scaled up to screen hundreds of compounds. The flexibility that this assay provides makes it a good first choice to test if a compound or a series of compounds may play a role in angiogenesis.

Angiogenic laminin-derived peptides stimulate wound healing.

Acceleration of the wound healing process by using angiogenic peptides has been demonstrated previously. Here we used select laminin-111 peptides, A13 and C16, from the laminin alpha1 and gamma1 chain, respectively, to test whether they are able to stimulate wound healing in a rat full thickness wound model. The 12-mer peptides C16 and A13 are highly angiogenic and bind to integrins alphavbeta3 and alpha5beta1. We show that A13 increases wound re-epithelialization as much as 17% over controls by day 4 and C16 increases coverage by 11%. Contraction of the treated wounds was increased as much as 11% for A13 and 8% for C16 at day 4. No differences were observed at day 7 with either peptide. The peptides also stimulated fibroblast migration in Boyden chamber assays. A13 increased cell migration as much as 2.4-fold on uncoated filters and as much as 16-fold on collagen type IV-coated filters over negative controls. Similarly, C16 also stimulated migration 1.8-fold on uncoated filters and as much as 12-fold on collagen-coated filters. A13 and C16 significantly decreased expression of the pro and active forms of matrix metalloproteinase 2 in foreskin fibroblasts indicating their role in collagen accumulation. We conclude that small bioactive angiogenic peptides can promote dermal wound healing and may offer a new class of stable and chemically manipulable therapeutics for wound healing.

Angiogenesis inhibition and choroidal neovascularization suppression by sustained delivery of an integrin antagonist, EMD478761.

PURPOSE: To evaluate the angiogenic inhibitory effects of an alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist, EMD478761, released from a polymeric implant in a chick chorioallantoic membrane (CAM) assay and laser-induced experimental choroidal neovascularization (CNV) in rats. METHODS: Polyvinyl alcohol-based reservoir implants releasing EMD478761 were designed for placement onto a CAM or intravitreally in rats. In vitro release rates of the implants were measured using HPLC. Angiogenesis was induced on 10-day-old chick embryos by basic fibroblast growth factor (bFGF), and areas of neovascularization were measured. Experimental CNV was induced in the Brown-Norway rat with a diode laser. EMD478761 or sham microimplants were placed within the vitreous chamber of Brown-Norway rats. Two weeks later, areas of CNV were determined by FITC-dextran staining of choroidal flatmounts. RESULTS: Sustained delivery of EMD478761 significantly inhibited bFGF-induced angiogenesis in CAM, as determined by a reduction in angiogenesis areas, without drug toxicity to the normal CAM vasculature. In an experimental rat model, intravitreal EMD478761 implants significantly suppressed laser-induced CNV compared with intravitreal sham implants, with the mean area reduced by 63% (P < 0.05). CONCLUSIONS: Sustained delivery of EMD478761demonstrates potent antiangiogenic properties in vivo. These results suggest that an EMD478761 implant may be beneficial in the treatment of neovascular ocular diseases.

Identification of a potent peptide antagonist to an active laminin-1 sequence that blocks angiogenesis and tumor growth.

The extracellular matrix plays an important role in many physiological processes. We have identified >20 angiogenic sites in the extracellular matrix protein laminin-1. The most potent sites are A13 (RQVFQVAYIIIKA) and C16 (KAFDITYVRLKF), which are present in homologous NH(2)-terminal domains of the alpha 1 and gamma 1 chains, respectively. We reported recently that a scrambled C16 sequence, C16S (DFKLFAVTIKYR), acts as an antagonist to both peptides. Here, we have identified a stronger antiangiogenic peptide, C16Y (C16S with a T to Y substitution), with potent activity in several biological assays including tumor growth. C16Y is more potent in promoting endothelial cell attachment and inhibiting attachment to laminin-1 than either C16 or C16Y. Disruption of tube formation by C16Y is also observed at concentrations at least five times lower than C16S. The minimal active sequence was found to be DFKLFAVY. C16Y is more potent in blocking C16-induced chick chorioallantoic membrane angiogenesis than C16S. Tumor growth studies on the chick chorioallantoic membrane showed that C16Y reduces breast cancer cell growth without affecting cell proliferation. This result suggests that angiogenesis is being inhibited by the peptide. In vivo animal studies demonstrated that C16Y treatment significantly reduced tumor growth and decreased tumor vessel number, as compared with controls, additionally suggesting that angiogenesis was affected. These results indicate that we have identified a more potent antiangiogenesis inhibitor peptide that may be used as a therapeutic to treat cancer.

Identification of redundant angiogenic sites in laminin alpha1 and gamma1 chains.

The degradation of the extracellular matrix is one of the first steps involved in angiogenesis, the formation of new vessels from preexisting ones. Laminin, a large extracellular matrix protein, has many biological activities, including the promotion of angiogenesis. Screening of the laminin-1 chains identified 20 angiogenic peptides, of which, A13 and C16, from the alpha1 and gamma1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins alpha5beta1 and alphavbeta3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the alpha1 chain. The peptides are located in homologous sites present in the last globular domains of their respective chains, and their amino acids are 66% conserved, as compared to the inactive homologous site in the beta1 chain, B19 to B20, which is only 18%-23% conserved. Cell attachment studies demonstrated that both A13 and C16 reciprocally inhibited their adhesion activity, whereas the corresponding laminin beta1 chain peptides were inactive. Chorioallantoic membrane assays showed that the in vivo angiogenic activity of A13 is blocked by a C16 antagonist, C16S, which also binds to the same integrin receptors. A13 affinity chromatography and immunoprecipitation analysis showed that the alphavbeta3 and alpha5beta1 integrin receptors bind to this sequence. We have therefore identified redundant activity on two laminin chains. These highly conserved functional sites are likely important mediators of the biological responses of laminins because either one or both of these chains (active sites) are present in almost all laminin isoforms identified to date.

The chick chorioallantoic membrane as an in vivo angiogenesis model.

The chick chorioallantoic membrane (CAM) assay for angiogenic activity is a model originally developed to study the angiogenic activity of tumor samples. It is an in vivo assay that can be readily performed in any laboratory setting. The effects of a test compound on angiogenesis are tested by exposing day 10 embryos to the compound and following the patterns of blood vessel development, scoring the appearance of the CAM at day 12 or 13.