Masayo Aoki

siRNA Knockdown of Tissue Inhibitor of Metalloproteinase-1 in Keloid Fibroblasts Leads to Degradation of Collagen Type I

Keloids are defined as overgrowths of scar tissue resulting from abnormal wound healing. They are characterized by excessive dermal deposition of thick, hyalinized collagen bundles resulting from an imbalance between the production and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are two important regulators of ECM degradation and remodeling. To evaluate the role played by knockdown of TIMPs in keloid formation, we transduced human keloid-derived fibroblasts (KFs) with small interfering RNAs targeting TIMP-1 or -2 (siTIMP-1 or siTIMP-2) using a lentiviral vector and assessed the biological effects. We found that MMP-1/TIMP-1 and MMP-1/TIMP-2 complexes were suppressed and that MMP-2 activity was upregulated in KFs expressing siTIMP-1 or siTIMP-2. In addition, increased degradation of collagen type I was observed in the supernatant of KFs expressing siTIMP-1, but not siTIMP-2, with the suppression of cell viability and induction of apoptosis. These results suggest that targeting TIMP-1 using small interfering RNA has significant therapeutic potential as an approach to treating keloids through degradation of their thick collagen bundles.

Tissue Inhibitor of Metalloproteinase-2 Suppresses Collagen Synthesis in Cultured Keloid Fibroblasts

Background: Keloids are defined as a kind of dermal fibroproliferative disorder resulting from the accumulation of collagen. In the remodeling of extracellular matrix, the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is as critical as the proper production of extracellular matrix. We investigate the role of TIMPs and MMPs in the pathogenesis of keloids and examine the therapeutic potential of TIMP-2. Methods: The expression of TIMPs and MMPs in most inflamed parts of cultured keloid fibroblasts (KFs) and peripheral normal skin fibroblasts (PNFs) in the same individuals and the reactivity of KFs to cyclic mechanical stretch were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (n = 7). To evaluate the effect of treating KFs with TIMP-2, collagen synthesis was investigated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and microscopic analysis was used to examine the treatment effects of TIMP-2 on ex vivo cultures of keloid tissue (n = 6). Results: TIMP-2 was downregulated in cultured KFs compared with PNFs in the same individuals, and the reduction in TIMP-2 was exacerbated by cyclic mechanical stretch. Administration of TIMP-2 (200 or 300 ng/mL) significantly suppressed expression of Col1A2 and Col3A1 mRNA and collagen type I protein in KFs. TIMP-2 also significantly reduced the skin dermal and collagen bundle thickness in ex vivo cultures of keloid tissue. Conclusion: These results indicated that downregulation of TIMP-2 in KFs is a crucial event in the pathogenesis of keloids, and the TIMP-2 would be a promising candidate for the treatment of keloids.

Experimental Rat Skin Flap Model That Distinguishes between Venous Congestion and Arterial Ischemia: The Reverse U-Shaped Bipedicled Superficial Inferior Epigastric Artery and Venous System Flap

Background: The commonly used flap models have drawbacks that limit their usefulness. In the random skin flap model, flap necrosis is caused by both arterial and venous insufficiency. In the axial skin flap model, flap viability is easily affected by the pedicle blood flow and can result in complete necrosis. This study aimed to establish a new rat skin flap model that has a consistent flap survival rate and in which venous congestion and arterial ischemia can be readily distinguished macroscopically. Methods: Rats underwent reverse U-shaped bipedicled superficial epigastric artery flap elevation. The right superficial epigastric vessels formed the pedicle. In the control rats (n = 3), the left superficial epigastric vessels were left intact. In the ischemia group (n = 10), the left superficial epigastric artery was ligated. In the congestion group (n = 10), the left superficial epigastric vein was ligated. The flap was returned to the original site and sutured. The surrounding neovascularization was blocked by polyurethane film. Flap survival rates were evaluated on postoperative day 3. Results: The flaps in the ischemia and congestion groups were noticeably pale and violet, respectively. Flap necrosis was noted in the contralateral distal zone only. It started on postoperative day 2 in the ischemia and congestion groups. The mean flap survival rates of the control, ischemia, and congestion groups were 100 percent, 61.8 percent (range, 56.9 to 67.1 percent), and 42.3 percent (35.7 to 48.7 percent), respectively (all p < 0.001). Conclusions: The flap facilitated discrimination of the effects of ischemia and congestion. This new rat skin flap model is simple and easy to construct, and has a consistent flap survival rate.

Chest wall reconstruction of severe mediastinitis with intercostal artery-based pedicled vertical rectus abdominis muscle flap with oblique-designed skin pedicle.

Recurrent poststernotomy mediastinitis has significant morbidity and mortality. Reconstructive treatment begins with pectoral muscle or omental flaps. When these options are unavailable or inadequate, surgeons resort to internal mammary artery-based vertical rectus abdominis muscle flap. If the internal mammary artery is harvested for coronary artery bypass grafting, surgeons are reluctant to use the muscle for pedicled flap because of the elevated risks. However, recent reports suggest that if enough time passes for intercostal artery collaterals to develop, they would support the viability of the flap. Moreover, recent improvements in defining flap microvasculature and proposed surgical techniques have enabled us to further refine the procedure. Although it appears that the intercostal artery-based pedicled vertical rectus abdominis muscle flap with oblique-designed skin pedicle is safe and effective for chest wall reconstruction, potential for failure remains elevated until sample size accumulates.

Histological analysis of hyalinised keloidal collagen formation in earlobe keloids over time: collagen hyalinisation starts in the perivascular area

Keloids grow and do not regress. They are characterised histologically by hyalinised keloidal collagen (HKC). HKC amounts vary, and the mechanism by which they form is unclear. To clarify how HKCs form and whether their formation associates with specific clinical features, we studied the histological findings of earlobe keloids and compared them with respective clinical features. A total of 50 earlobe keloids from 43 patients were used for histological analysis of keloid size (mm2), HKC area (mm2) and HKC area ratio (%). As a result, keloid durations ranged from 3 months to >13 years. Early‐stage keloids exhibited little HKC and a tendency for the HKCs to locate in perivascular regions. In later‐stage keloids, the HKCs were extremely interconnected and formed a thick bitten donut‐shaped region. HKC area ratios correlated positively with keloid duration (r2 = 0·58, P<0·05). HKC area ratios and keloid durations did not correlate with keloid sizes. These patterns of HKC formation and growth may explain why local therapies, which effectively remove fibroblasts and accumulated collagen but not HKCs, are ineffective in older keloids. Keloids should be promptly treated after diagnosis, and older keloids with extensive HKCs may require surgical excision followed by radiotherapy.

Objective Spectrometric Measurement of Keloid Color in the East Asian Population: Pitfalls of Subjective Color Measurements

BACKGROUND Keloids are characterized by the formation of excessive scar tissue that extends beyond the area of the initial wound. Keloid redness is due to angiogenesis and chronic inflammation and is an important indicator of the severity of the lesion and the effectiveness of treatment. METHODS The color of 33 untreated keloids from 30 patients was measured with a narrow-band reflectance colorimeter. The erythema and melanin levels in the keloids (Ek and Mk, respectively) were recorded with control data obtained from the flexor aspect of the forearm (Ec and Mc, respectively). The keloid color was also evaluated subjectively. RESULTS The Ek or Mk values did not vary significantly according to symptom intensity, scar region, patient age, and patient sex. Younger patients (<40 years) and female patients had significantly higher Ek/Ec ratios than did older patients and male patients, respectively. Subjective keloid redness evaluations distinguished keloids with high Ek/Ec ratios from keloids with low Ek/Ec ratios (P<0.0001) but could not distinguish keloids with high Ek from keloids with low Ek. CONCLUSIONS Subjective evaluations of keloids in Japanese subjects reflected Ek/Ec ratios, which were strongly affected by variation in background skin color. The subjective assessment of the color of keloids or other skin disorders should be performed with caution in Asian populations.