Keloidal Collagen May Be Produced Directly by αSMA-positive Cells: Morphological Analysis and Protein Shotgun Analysis

Abstract

Keloids are fibroproliferative lesions caused by abnormal dermal wound healing. Keloidal collagen (KC) is a pathognomic feature of keloids, but the mechanism by which it forms is unknown. This study aimed to evaluate the histopathology of KC and thereby gain clues into how it forms.

Methods: The cross-sectional study cohort consisted of a convenience series of patients with keloids who underwent surgical excision. Skin pieces (3 mm²) were collected from the keloid center and nearby control skin. Histopathology was conducted with light and electron microscopy and immunohistochemistry. KC composition was analyzed with protein shotgun analysis.

Results: Microscopic analyses revealed the ubiquitous close association between KC and αSMA-positive spindle-shaped cells that closely resembled myofibroblasts. Neither KC nor the spindle-shaped cells were observed in the control tissues. Compared with control skin, the collagen fibers in the KC were overall thinner, their diameter varied more, and their spacing was irregular. These features were particularly pronounced in the collagens in the vicinity of the spindle-shaped cells. Protein shotgun analysis did not reveal a specific collagen in KC but showed abnormally high abundance of collagens I, III, VI, XII, and XIV.

Conclusions: These findings suggest that KC may be produced directly by myofibroblasts rather than simply being denatured collagen fibers. Because collagens VI and XII associate with myofibroblast differentiation, and collagen XIV associates with local mechanical stress, these collagens may reflect, and perhaps contribute to, the keloid-specific local conditions that lead to the formation of KC.

Fig. 1.

Collection of keloid tissues. A representative keloid that was excised by surgery is shown. The 3-mm² pieces of skin that were collected from the center of the keloid (a) and the control skin near the keloid (b) are shown.

Fig. 2.

Light microscopy image of toluidine blue– and eosin-stained keloid tissue in cross-section. A representative image of the 13 keloid pieces is shown in cross-section. The KC is the red streak–like hyalinized areas in the reticular layer of the dermis.

Fig. 3.

Light microscopy image of toluidine blue–stained keloid tissue in cross-section. A representative image of the keloid pieces is shown in cross-section at 100-µm magnification. The KC in the reticular dermis is interspersed with new blood vessels (★) and infiltrating cells (↑), including mast cells (▲).

Fig. 4.

TEM images of keloid tissue. A representative image of the keloid pieces is shown in cross-section at 20-µm (A), 5-µm (B), and 1-µm (C and D) magnification. In A, the KC is marked by KC, the blood vessels by ★, and the infiltrating cells by ↑. The mast cells are indicated by ▲. The image in B is a magnification of the area near the cells in A. The areas in B that are magnified in C and D are indicated by the inset borders. Spindle-shaped cells can be seen. In C and D, KC is indicated by KC, fibronexus by FNX, fibronectin filaments by ff, myofilaments by mf, and dense body by db.

Fig. 5.

Immunohistochemistry image of αSMA expression in the KC area. A representative of the keloid pieces that underwent immunohistochemistry for αSMA expression is shown in cross-section at 50-µm magnification. A, Immunostaining for αSMA. B, The negative control lacking the anti-αSMA antibody.

Fig. 6.

TEM images of the collagen fibers in the KC area. Cross-section images are shown to the left, and longitudinal section images are shown to the right. Upper panels: Collagen fibrils around the cells in the KC area. Lower panels: Collagen fibrils in the control skin.

Fig. 7.

Box plot showing comparison of collagen fibril diameters in the central KC area and control skin. The box line is the median, and the IQR is the difference between the third and first quartile. (***P < 0.001 by Mann-Whitney U test.).

Fig. 8.

Shotgun protein analysis of KC. The KCs of three keloids were microdissected and subjected to protein shotgun analysis followed by comparative quantification of the identified proteins by iBAQ values. The three keloid samples contained 500, 621, and 552 factors. They shared 371 factors. The 10 most abundant collagen factors are shown in the table. The relative abundances (log iBAQ) of the ten collagen factors were plotted.