M. Lourdes Ponce

Identification of laminin α1 and β1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting

Laminin‐1 is a basement membrane glycoprotein that promotes several biological activities including cell attachment, tumor metastasis, and angiogenesis. Angiogenesis plays an important role in tissue formation, reproduction, wound healing, and several pathological conditions. In this study, we screened 405 synthetic peptides from the α1 and β1 chains to identify potential sites on laminin‐1 active with endothelial cells. Peptides were initially screened by testing both endothelial cell adhesion to peptide‐coated wells and tube formation on Matrigel in the presence of soluble peptide. Twenty active peptides were identified in these screens. A secondary screen using the rat aortic ring sprouting assay identified 13 of the 20 peptides that stimulated endothelial sprouting. Several of these active peptides were also found to stimulate human umbilical vein endothelial cell migration in Boyden chamber assays. Differences in the amount of peptide needed for the response and in the resultant morphologies/responses were observed between the peptides in all of the assays. Our results suggest that several active domains on laminin‐1 may play important roles in stimulating different steps in angiogenesis.—Malinda, K. M., Nomizu, M., Chung, M., Delgado, M., Kuratomi, Y., Yamada, Y., Kleinman, H. K., Ponce, M. L. Identification of laminin α1 and β1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting. FASEB J. 13, 53–62 (1999)

Identification of Endothelial Cell Binding Sites on the Laminin γ1 Chain

Abstract—The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like struc...

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.

An angiogenic laminin site and its antagonist bind through the αvβ3 and α5β1 integrins

Angiogenesis is important for wound healing, tumor growth, and metastasis. Endothelial cells differentiate into capillary‐like structures on a laminin‐1‐rich matrix (Matrigel). We previously identified 20 angiogenic sites on laminin‐1 (α1β1γ1) by screening 559 overlapping synthetic peptides. C16, the most potent γ1 chain peptide, blocked laminin‐1‐mediated adhesion and was the only γ1 chain peptide to block attachment to both collagen I and fibronectin. This suggested that C16 was acting via a receptor common to these substrates. We demonstrated that C16 is angiogenic in vivo. Affinity chromatography identified the integrins α5β1 and avβ3 as surface receptors. Blocking antibodies confirmed the role of these receptors in C16 adhesion. C16 does not contain an RGD sequence and, as expected, an RGD‐containing peptide did not block C16 adhesion nor did C16 act via MAP kinase phosphorylation. Furthermore, we identified a C16 scrambled sequence, C16S, which antagonizes the angiogenic activity of bFGF and of C16 by binding to the same receptors. Because the laminin γ1 chain is ubiquitous in most tissues, C16 is likely an important functional site. Since the biological activity of C16 is blocked by a scrambled peptide, C16S may serve as an anti‐angio‐genic therapeutic agent.—Ponce, M. L., Nomizu, M., Kleinman, H. K. An angiogenic laminin site and its antagonist bind through the avβ3 and α5β1 integrins. FASEB J. 15, 1389–1397 (2001)

In Vitro Matrigel Angiogenesis Assays

A variety of in vivo and in vitro methods have been used to study angiogenesis, the process of blood vessel formation. Two widely accepted but technically difficult assays include the cornea implant assay and the chick chorioallantoic membrane assay. The cornea assay requires special equipment and a skilled person to implant beads containing the test compound in the eyes of animals; only a small number of samples can be tested due to cost and time. The chorioallantoic membrane assay requires a large number of samples on account of the variability of the system and its difficulty in quantitation. In our laboratory, we have developed a quick and highly reliable method for testing numerous compounds for angiogenic and/or antiangiogenic activity. The method is based on the differentiation of ECs on a basement membrane matrix, Matrigel, derived from the Engelbreth-Holm-Swarm tumor (1). ECs from human umbilical cords as well as from other sources differentiate and form capillary-like structures on Matrigel in the presence of 10% bovine calf serum (BCS) and 0.1 mg/mL of endothelial cell growth supplement (ECGS) (2), which is a mixture of both acidic and basic fibroblast growth factor (Fig. 1, Panel C). The formation of tube-like vessels under these conditions can be used to assess compounds that either inhibit or stimulate angiogenesis.

Identification of redundant angiogenic sites in laminin α1 and γ1 chains

Abstract 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 α1 and γ1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins α5β1 and αvβ3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the α1 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 β1 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 β1 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 αvβ3 and α5β1 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.

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.

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.