Angiogenic laminin-derived peptides stimulate wound healing

Abstract

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.

Figure 1

Laminin-derived peptides increased reepithelialization when applied topically. A) Diagram of a full thickness punch wound. The reepithelialization as measured by % coverage and contraction as measured by % granulation were used to calculate wound closure. % reepithelialized= Tongue + Tongue /Total Wound Length ×100. % granulated = 1- Distance Between Migrating Dermal Fronts/Total Wound Length ×100 B) Quantitation of wound reepithelialization in wounds harvested at day four. Wounds were treated with peptides ranging from 2.5 to 50 µg in 50 µl. Treatment with A13 at the 2.5 and 5 µg in 50 µl level, wounds showed a significant increase in the increase in epithelial coverage of the wound, 7% (±2.5) and 17% (±3.8), respectively. At the higher dose of 50 µg in 50 µl, A13 showed a statistically significant level of inhibition of reepitheilization at 12% (±3.8). C16 peptide treatment also increased reepithelialization in topical treatments. At all doses, a significant increase in the levels of reepithelialization was observed. At 2.5 µg, a 10% (±2.9) increase in coverage was observed. At 5 µg, coverage increased to 11% (±2.4) and at higher doses, 25 µg in 50 µl, an 8% (± 2.2) increase in coverage was observed. Measurements are expressed as the mean % increase ± SEM. (^*p≤0.008, ^**p≤0.02).

Figure 2

Treatment with laminin-derived peptides enhanced granulation when applied topically. Wounds were harvested at day 4. Wounds were treated with 2.5 to 50 µg in 50 µl. A13 treatments showed significant increases in granulation. At 2.5 µg, A13 showed a 6% (± 2.6) increase. Granulation increased at the 5 µg dose to 11% (±3). At higher doses of A13, a decrease was observed. At 25 µg, an 11% (±3) decrease in granulation was observed. The effect at the 50 µg dose was not statistically significant. C16 treatments resulted in 9% (±3) increase in granulation at 2.5 µg and 5 µg doses. A slightly lower increase of to 8% (±3) occurred at the 25 µg dose. Measurements are expressed as the mean % increase ± SEM. (^*p≤0.002, ^**p≤0.02).

Figure 3

A: Comparison of laminin-derived peptides and control on wound healing at day seven. Dosages of 2.5 and 5 µg in 50 µl of A13 and C16 were tested. Only A13 at the 2.5 µg dose showed a 10% (± 5) inhibition of reepithelialization. Other treatment had no significant effect. B. Treatment with peptides at the 2.5 and 5 µg in 50 µl doses had no significant effect on granulation. Treatment with A13 and C16 had no statistically significant difference in the granulation of the wounds. Measurements are expressed as the mean % increase ± SEM. (^*p=0.048)

Figure 4

Laminin peptides enhanced wound healing in rats. Masson’s Trichrome stained sections of 4-day punch wounds show collagen in blue and endothelial cells in red: Arrows on Panels A, D and G indicate the edge of the original wound. Boxed areas of the wound show areas magnified on the following image. A) Wound treated with laminin-derived peptide A13 (5 µg/50 µl). Epithelial tongues are visible migrating over the dermis as reepithelialization proceeds. B–C) Granulation tissue is infiltrated with cells and a moderate number of vessels (see arrows). Enhanced collagen production was present in the treated sections (dark blue staining fibers). D) Wound treated with laminin peptide C16 (5 µg/50 µl). E–F) Here areas of granulation show neovascularization (see arrows) and enhanced numbers of fibroblasts. Collagen levels also appear elevated in the treated wounds (blue staining fibers). G) Vehicle control. H–I) Control shows few cells and little vascularization (see arrow). Magnification: A,D,G: (0.96x), B,E,H: 2.4x, C,F,I: 4.8x bar size: 1 mm

Figure 5

Foreskin fibroblast migration was stimulated in the presence of laminin peptides. A) A13 in the lower wells of a Boyden chamber stimulated migration above background levels on uncoated filters. There was a 1.6- to 2.5-fold increase in migration above baseline depending on the concentration of peptide tested. C16 peptide tested in the Boyden chamber also stimulated migration above baseline levels. A 1.3- to 1.8-fold increase was seen in migration on uncoated filters. (^*p≤0.009, ^**p≤0.04), B) Migration of foreskin fibroblasts on collagen-coated filters was enhanced. A13 added to the bottom well of the Boyden chamber caused a 15- to 16-fold increase in migration at the lower concentrations, however, tests at higher concentrations resulted in lower fold increases or at the highest a decrease in migration. C16 treatment of the media in the lower well caused an approximate 7- to 12-fold increase in migration. For this experiment all background levels of migration were subtracted. (^*p≤0.004, ^**p≤0.015).

Figure 6

Peptide treatment decreases MMP-2 activity. A) Representative zymogram of media collected from HUVECs cultured with for 18 hrs with 5 and 10 µg/ in 50 µl of A13 or C16. Pro-MMP-2 levels were slightly decreased over control levels at the 5 and 10 µg/50 µl doses of C16 and at the dose 10 µg/50 µl of A13. Processed MMP-2 was decreased as compared to controls at the 5 and 10 µg/50 µl doses of C16 and steadily decreased as the concentration of the peptide A13 was increase. This occurred in two of three cases. B) Representative zymogram of media collected from foreskin fibroblasts cultured for 18 hrs with 2.5, 5, and 10 µg/ in 50 µl of A13 or C16. Treatment with A13 decreased MMP-2 levels below background even at the lowest concentration tested. C16 treatment showed an incremental decrease of MMP levels with trace amounts of MMP detectable at the highest concentration. This result was repeated 3 times.