Specific effects of fibrinogen and the γA and γ'-chain fibrinogen variants on angiogenesis and wound healing

Abstract

In a newly formed wound, the natural fibrin network provides the first temporary matrix for tissue repair. Topical application of fibrin to a new wound may improve wound healing. A matrix of the common natural γ' fibrin variant may further improve wound healing because it is expected to have a different architecture and this will influence angiogenesis, because it possesses increased thrombin and factor XIII binding and decreased platelet binding, when compared with the common γA fibrin matrix. Our objective was to determine the effect of fibrinogen and its γA and γ' variants on angiogenesis and wound healing. We used in vitro angiogenesis models and an in vivo rat full-thickness excisional wound healing model. When comparing γA and γ' fibrin in vitro, more tube-like structures were formed on day 7 in γA fibrin than in γ' fibrin (13.83±6.12 AU vs. 6.1±1.46 AU). Wounds treated with fibrin demonstrated improved healing in vivo with more perfusion (47%±3% vs. 26%±4%, p<0.01 in placebo) and higher CD34 density score (2.0±0.4 vs. 2.8±0.1, p<0.01) on day 21 with fibrin matrices when compared with placebo-treated wounds. Increased perfusion was observed in γA fibrin-treated wounds on day 21 (53%±10% vs. 41%±7% for γ' fibrin). The other parameters showed slightly improved (not significant) wound healing with γA fibrin compared with γ' fibrin matrices. In conclusion, the use of fibrin and fibrin variant matrices offers an interesting methodology to stimulate the wound healing process.

FIG. 1.

Characterization of the γA and γ′ fibrinogen fractions. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis with total protein staining and (B) western blotting of fibrinogen with polyclonal human fibrinogen antibody (red) and monoclonal human γ′ fibrinogen antibody (green). M, protein electrophoresis marker. Color images available online at www.liebertpub.com/tea

FIG. 2.

Confocal microscopy of γA and γ′ fibrin matrices. Fibrin matrices prepared from γA fibrinogen without additional FXIII (γA), γA supplemented with FXIII (γA+FXIII), and γ′ fibrin (γ′). The scale bars indicate 5 μm. Color images available online at www.liebertpub.com/tea

FIG. 3.

In vitro functional characteristics of human umbilical vein endothelial cells (HUVECs) on various fibrinogen coatings. (A) Adhesion of HUVECs to gelatin (solid line), γA fibrinogen (dashed line), γ′ fibrinogen (dotted line), and 1% bovine serum albumin (dashed-dotted line) coatings (n=3). (B) The proliferation of HUVECs on gelatin, γA, and γ′ fibrinogen coatings, measured by ³H-thymidine incorporation (n=3). The values were corrected for cell adhesion and expressed as percentage of HUVEC proliferation on gelatin coatings. (C) The migration of HUVECs on gelatin, γA, and γ′ fibrinogen coatings; after 16 h of migration, the area of migration was determined (n=6).

FIG. 4.

Representative images of endothelial cell migration, based on the in vitro assay. The length of the bar is 0.5 mm.

FIG. 5.

In vitro tube formation by human microvascular endothelial cells (HMVECs). HMVECs were seeded on three-dimensional γA (A, C) and γ′ fibrin (B, D) matrices and tubular structure formation was stimulated. Representative pictures of the in vitro tube formation assay, using light microscopy with visible tube-like structures are depicted with black arrows (A, B) and fluorescent staining where white arrows depict tube-like structures (C, D). The fluorescent staining represents the F-actin filaments of endothelial cells; the bright structures represent the tubular structures in the fibrin matrix. In γA fibrin many structures are present, only structures in the left-lower corner are depicted. An enlargement of figure (C, E) is given. Z-stacks confirmed the three-dimensional nature of the tube-like structures. (E) Quantification of the amount of tube formation in the various fibrin matrices (n=3). (F) Fibrin degradation products that were present in conditioned medium of the in vitro tube formation assay, as determined by enzyme-linked immunosorbent assay (n=3). *p<0.05. Color images available online at www.liebertpub.com/tea

FIG. 6.

Revascularization of full-thickness rat wounds treated with fibrinogen or placebo on days 7 and 21. The symbol □ gives data of placebo-treated wounds (n=6) and \\\ fibrin-treated wounds (n=17). *p<0.05.

FIG. 7.

Representative images of in vivo wound closure of the wounds. Color images available online at www.liebertpub.com/tea

FIG. 8.

Representative images of the CD34 immunohistochemistry staining of control wounds and wounds treated with unfractionated, γA fibrin, and γ′ fibrin at day 21. Color images available online at www.liebertpub.com/tea

FIG. 9.

Inflammation score in full-thickness rat wounds on day 21 (blank n=6, unfractionated n=5, γA fibrin n=6, and γ′ fibrin n=6). *p<0.05.