Effects of Recombinant Angiogenin on Collagen Fiber Formation and Angiogenesis in the Dermis of Wistar Rats

Abstract

Purpose: The purpose of this study was to assess the capability of recombinant angiogenin isolated from Pichia pastoris yeasts to stimulate regenerative processes in the dermis of experimental animals.

Patients and methods: Wistar rats were administered with recombinant angiogenin intracutaneously. Morphological examination of the skin and the assessment of the proliferative activity of the epidermal cells were carried out. Additionally, cytokine production by human whole blood cells exposed to angiogenin was analyzed ex vivo.

Results: Administration of angiogenin stimulates collagen fiber formation and angiogenesis. This stimulation is tightly associated with an increase in the number of fibroblasts, an increased numerical density of dermal blood vessels and an increased density of collagen fibers; also, it activates the proliferation of basal cells. Angiogenin induces the production of MCP, IL-8, IL-6, IL-1β, TNF-α, IL-10, TGF-β, and VEGF by blood cells.

Conclusion: The results obtained indicate a broad spectrum of actions of recombinant angiogenin during regenerative processes in the basal layer of the dermis.

Keywords: cytokines; intracutaneous injections; pathomorphological analysis; regeneration.

Figure 1

Skin from the back of the intact animal and animals after three and six injections of each saline, 0.4 and 0.8 μg of angiogenin. Expansion of the stratum corneum, an increase in the number of epidermal layers, thickened collagen fibers, the site of newly formed collagen with proliferating fibroblasts (the edge of the injection area). Hematoxylin/eosin staining, 100× magnification.

Figure 2

Quantification of pathomorphological parameters in the dermis of the intact animal and animals treated three or six times with each saline, 0.4 and 0.8 μg of angiogenin. (A) Thickness of the dermis (μm); (B) thickness of the stratum corneum (μm); (C) number of fibroblasts, (D) number of blood vessels; (E) density of elastic fibers; (F) density of collagen fibers in the treatment area. The method for calculating these parameters is given in the Materials and Methods section. *Significant differences from the intact controls (p<0.05), ^#significant differences from the saline-treated group (p<0.05).

Figure 3

Proliferation of cells of the epidermis from the back of animals. (A) Ki-67-positive cells (%). *Significant differences from the intact controls (p<0.05), ^#significant differences from the saline-treated group (p<0.05). (B) Immunohistochemical staining of skin sections from animals after three and six injections of saline, 0.4 and 0.8 μg of angiogenin. Arrows point to the stained nuclei of basal cells of the epidermis. 200× magnification.

Figure 4

Effects of angiogenin at doses of 0.01, 0.1 and 1 μg/mL on cytokine production by whole blood cells from healthy donors (n = 3). The y-axis represents the “Influence Index” (the ratio of cytokine production by the cells exposed to angiogenin at the corresponding concentration to the basal level of cytokine production by untreated cells). The red line signifies the basal level of cytokine production (taken as “1”).