Katherine M. Malinda

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells.

Thymosin β4 (Tβ4) is a 4.9 kDa polypeptide that interacts with G‐actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. Tβ4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of Tβ4 in endothelial cell migration. Our results demonstrate that Tβ4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four‐ to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to Tβ4 treatment, suggesting that the migration activity of Tβ4 was endothelial cell‐specific. Tβ4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. Tβ4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that Tβ4 stimulated cell migration in vivo. These results provide the first direct evidence that Tβ4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.—Malinda, K. M., Goldstein, A. L., Kleinman, H. K. Thymosin β4 stimulates directional migration of human umbilical vein endothelial cells. FASEB J. 11, 474–481 (1997)

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...

In Vivo Matrigel Migration and Angiogenesis Assays

Angiogenesis, the process of new blood vessels forming from preexisting vessels, is an important feature in developmental processes, wound healing, and pathologic conditions such as cancer and vascular diseases. Owing to the importance of angiogenesis, a relatively simple and rapid in vivo method to determine the angiogenic potential of compounds is desirable to augment in vitro findings.

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.

Role of Thymic Peptides in Wound Healing

Wound repair requires the concerted action of numerous cells and factors. The inflammation, proliferation, and remodeling phases of wound healing occur in a highly coordinated cascade. Angiogenesis, the formation of new blood vessels, is one of the critical steps in the proliferative phase of wound repair (1–5). Like wound healing, angiogenesis requires cell migration, proliferation, and extracellular matrix synthesis and assembly. New vessels provide nutrients to support the repair cells, promote granulation tissue formation, and facilitate the clearance of debris. Granulation tissue is mainly composed of blood vessels that also supply the necessary oxygen to stimulate repair. Wound angiogenesis is a complex multipstep process that involves many mediators. Despite a detailed knowledge about many angiogenic factors present in the wound, little progress has been made in defining the source of these factors and the regulatory events involved in their production (1–9). Further complicating the understanding of wound angiogenesis and repair is the fact that the mechanisms and mediators involved in repair likely vary depending on the depth of the wound, type of wound (burn, trauma, etc.), and the location (muscle, skin, bone, etc.). The condition and age of the patient (diabetic, paraplegic, on steroid therapy, elderly vs infant, etc) can also determine the rate of repair and response to angiogenic factors. Furthermore, the sex of the patient and hormonal status (premenopausal, postmenopausal, etc.) may also influence the repair mechanisms and responses. Impaired wound healing particularly affects the elderly and many of the 14 million diabetics in the United States and reduced angiogenesis is often a causative agent for wound-healing problems in these patient populations.