Expressions of Collagen I and III in Hypoxic Keloid Tissue

Abstract

Background: Wound heals itself spontaneously as physiological process. However, in some individuals, small wounds such as parenteral injections or body piercings may cause increased expression of collagen synthesis. The condition is known as keloid. Histopathology of keloid demonstrates extensive tissue proliferation that extends beyond the margin of primary wound. As a result, it develops uncontrolled or excessive fibrogenesis and tremendous source of collagen that still causes clinical problems until now. A wound, no matter how small the size is, will be followed by increased expression of collagen synthesis. Procollagen I and III is one of markers indicating the development of fibrosis. In fibrosis, there is hypoxia, which is characterized by stabilization of HIF-1α. Therefore, our study was aimed to obtain information about expression of collagen I and III in hypoxic keloid tissue.

Method: The study design was observational descriptive. Keloid specimens were obtained from biopsy and preputium skins as the control specimens were obtained from circumcision. There were 10 tissue specimens for each specimen group. The analysis performed were evaluation of mRNA expression on collagen I, collagen III and HIF-1α using RT-PCR, the evaluation of HIF-1α protein level using ELISA and the expression of collagen I and collagen III protein using immunohistochemistry. Statistically, data was analyzed by unpaired t-test.

Results: In keloid with excessive cell proliferation, we found that the expression of procollagen I mRNA increased 35 times and the expression of procollagen III mRNA increased 27.1 times compared to preputium control group (p<0.05). The expression of procollagen I protein in the dermal layer of keloid was 61% and in the preputium was 37% (p<0.05). The expression of collagen III protein in the dermal layer of keloid was 39% and in the preputium was 16% (p<0.05). There was a 5-fold increase on expression of HIF-1α mRNA in keloid tissue compared to those in preputium (p<0.05). The levels of HIF-1α protein in keloid tissue was 0.201 ng/mg protein and the level in preputium was 0.122 ng/mg protein (p<0.05). There was a strong positive and extremely significant correlation between the expression of HIF-1α protein and procollagen III (R=0.744; p<0.05, Pearson), but HIF-1α with procollagen I are weak correlation (R=0.360; p>0.05, Pearson) Conclusion: Expression of collagen I and III have important role in hypoxic keloid tissue characterized by increased expressions. The expression of collagen I and III is associated with stable HIF-1α in keloid tissue.

Keywords: HIF-1α; Hypoxia; collagen; keloid.

Figure 1

Fibroblasts and collagen structure (arrows) are seen in keloid (A) and preputium (B) with hematoxylin-eosin staining (400 times magnification).

Figure 2

Expressions of collagen I mRNA and protein. (A) Ratio of procollagen I mRNA expression, in keloid tissue it was significantly higher than those in preputium tissue (*p<0.05). Expression of collagen I protein in the cells of dermal layer was found both in nucleus and cytoplasm characterized by brown staining (arrow, 400 and 1000 times magnifications). (C) Expression of collagen I in dermal layer cells of preputium tissue; (D) Expression of collagen I in dermal layer cells of keloid tissue; (E) Expression of collagen I in positive control tissue; (F) negative control. (B) A graph shows percentage ratio of the number of cells expressing collagen I protein in dermal layer of keloid and preputium tissues using immunohistochemistry and it shows significant difference (*p<0.05).

Figure 3

Expression of collagen III mRNA and protein. (A) Ratio of procollagen III mRNA expression in keloid tissue was significantly higher than those in preputium tissue (*p <0.05). The expression of collagen III protein in the cells of dermal layer was found both in nucleus and cytoplasm characterized by brown staining (arrow, 400 and 1000 times magnifications). (C) Expression of collagen III in dermal layer cells of preputium tissue; (D) Expression of collagen III in dermal layer cells of keloid tissue; (E) Expression of collagen III in positive control tissue; (F) negative control. (B) A graph shows percentage ratio of the number of cells expressing collagen III protein in dermal layer of keloid and preputium tissues using immunohistochemistry and it shows significant difference (*p<0.05).

Figure 4

mRNA expression and the level of HIF-1α protein. (A) A ratio of HIF-1α mRNA expression in keloid and preputium tissue using RT-PCR shows significant difference (*p<0.05). Data had been previously normalized to the control (B) A ratio of HIF-1α protein level in keloid and preputium tissue using ELISA technique shows significant difference (*p<0.05).

Figure 5

Correlation between HIF-1α protein level with expressing procollagen I (A) and procollagen III (B).

Figure 6

Biosynthesis of collagen. Biosynthesis of collagen stage include (i) the synthesis of procollagen in the reticulum endoplasmic (II) hydroxylation of proline and lysine (III) O-glycosylated hydroxylysine (IV) Three chain prokalogen are together and form a triple helix called monomers collagen (V and VI) the monomer collagen secreted from the cell (VII) termination propeptida (VIII) self-assemble into collagen fibrils and it is form crosslink covalent bonds between the monomer collagen (IX) aggregation Fibril.