Direct Delivery of Apatite Nanoparticle-Encapsulated siRNA Targeting TIMP-1 for Intractable Abnormal Scars.

Hypertrophic scars (HSs) and keloids are histologically characterized by excessive extracellular matrix (ECM) deposition. ECM deposition depends on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs). TIMP-1 has been linked to ECM degradation and is therefore a promising therapeutic strategy. In this study, we generated super carbonate apatite (sCA) nanoparticle-encapsulated TIMP-1 small interfering RNA (siRNA) (siTIMP1) preparations and examined the effect of local injections on mouse HSs and on ex vivo-cultured keloids. The sCA-siTIMP1 injections significantly reduced scar formation, scar cross-sectional areas, collagen densities, and collagen types I and III levels in the lesions. None of the mice died or exhibited abnormal endpoints. Apatite accumulation was not detected in the other organs. In an ex vivo keloid tissue culture system, sCA-siTIMP1 injections reduced the thickness and complexity of collagen bundles. Our results showed that topical sCA-siTIMP1 injections during mechanical stress-induced HS development reduced scar size. When keloids were injected three times with sCA-siTIMP1 during 6 days, keloidal collagen levels decreased substantially. Accordingly, sCA-siRNA delivery may be an effective approach for keloid treatment, and further investigations are needed to enable its practical use.

Gene Expression Profile of Isolated Dermal Vascular Endothelial Cells in Keloids.

Wound healing is a complex biological process, and imbalances of various substances in the wound environment may prolong healing and lead to excessive scarring. Keloid is abnormal proliferation of scar tissue beyond the original wound margins with excessive deposition of extracellular matrix (ECM) and chronic inflammation. Despite numerous previous research efforts, the pathogenesis of keloid remains unknown. Vascular endothelial cells (VECs) are a major type of inductive cell in inflammation and fibrosis. Despite several studies on vascular morphology in keloid formation, there has been no functional analysis of the role of VECs. In the present study, we isolated living VECs from keloid tissues and investigated gene expression patterns using microarray analysis. We obtained 5 keloid tissue samples and 6 normal skin samples from patients without keloid. Immediately after excision, tissue samples were gently minced and living cells were isolated. Magnetic-activated cell sorting of VECs was performed by negative selection of fibroblasts and CD45+ cells and by positive selection of CD31+cells. After RNA extraction, gene expression analysis was performed to compare VECs isolated from keloid tissue (KVECs) with VECs from normal skin (NVECs). After cell isolation, the percentage of CD31+ cells as measured by flow cytometry ranged from 81.8%-98.6%. Principal component analysis was used to identify distinct molecular phenotypes in KVECs versus NVECs and these were divided into two subgroups. In total, 15 genes were upregulated, and 3 genes were downregulated in KVECs compared with NVECs using the t-test (< 0.05). Quantitative RT-PCR and immunohistochemistry showed 16-fold and 11-fold overexpression of SERPINA3 and LAMC2, respectively. SERPINA3 encodes the serine protease inhibitor, α1-antichymotripsin. Laminin γ2-Chain (LAMC2) is a subunit of laminin-5 that induces retraction of vascular endothelial cells and enhances vascular permeability. This is the first report of VEC isolation and gene expression analysis in keloid tissue. Our data suggest that SERPINA3 and LAMC2 upregulation in KVECs may contribute to the development of fibrosis and prolonged inflammation in keloid. Further functional investigation of these genes will help clarify the mechanisms of abnormal scar tissue proliferation.

Sphingosine-1-Phosphate Facilitates Skin Wound Healing by Increasing Angiogenesis and Inflammatory Cell Recruitment with Less Scar Formation.

Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1-/- mice exhibit delayed wound closure along with less angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. The SphK1 overexpression also led to less scarring, and the interaction between transforming growth factor (TGF)-ß1 and S1P receptor-2 (S1PR2) signaling is likely to play a key role. In summary, SphK1 play important roles to strengthen immunity, and contributes early wound healing with suppressed scarring. S1P can be a novel therapeutic molecule with anti-scarring effect in surgical, trauma, and chronic wound management.

Cytochrome P450 genes play central roles in transcriptional response by keratinocytes to a high-voltage alternating current electric field.

The endogenous electric field (EF) of skin wounds plays an important role in the biological processes that underlie wound healing. Treatments that modulate wound-EFs promote healing. However, the mechanism(s) that underlie this effect remain unclear. Agilent-based microarrays were used to determine the transcriptomes of the keratinocyte line HaCaT, normal human dermal fibroblasts, and the human dermal endothelial cell line HMEC-1 before and after high-voltage alternating current (AC)-EF (14,000 V, 90 Hz) treatment. The keratinocytes had the most genes whose transcription was altered by EF. They included the cytochrome P450 (CYP) genes CYP1A1 and CYP1B1, HMOX1, EREG, DUSP5, and SLC7A11 (all upregulated), and DOCK8, ABCC6, and CYP26A1 (all downregulated). As shown by transcriptional-network analysis, all three CYP genes played central roles in the EF-induced changes in keratinocyte transcriptome. To the best of our knowledge, this is the first study that demonstrates that CYP genes play a key role in the transcriptional responses of human keratinocytes to EF treatment. Further investigations into the effects of EF on wound healing, aging, and regenerative medicine are likely to yield promising results.

Periauricular Keloids on Face-Lift Scars in a Patient with Facial Nerve Paralysis.

Keloids are caused by excessive scar formation that leads to scar growth beyond the initial scar boundaries. Keloid formation and progression is promoted by mechanical stress such as skin stretch force. Consequently, keloids rarely occur in paralyzed areas and areas with little skin tension, such as the periauricular region. Therefore, periauricular incision is commonly performed for face lifts. We report a rare case of keloids that arose from face-lift scars in a patient with bilateral facial nerve paralysis. A 51-year-old Japanese man presented with abnormal proliferative skin masses in bilateral periauricular scars. Seventeen years before, he had a cerebral infarction that resulted in permanent bilateral facial nerve paralysis. Three years before presentation, the patient underwent face-lift surgery with periauricular incisions. We diagnosed multiple keloids. We removed the masses surgically, closed the wounds with sutures in the superficial musculoaponeurotic system layer to reduce tension on the wound edges, reconstructed the earlobes with local skin flaps, and provided 2 consecutive days of radiotherapy. The wounds/scars were managed with steroid plasters and injections. Histology confirmed that the lesions were keloids. Ten months after surgery, the lesions did not exhibit marked regrowth. The keloids appeared to be caused by the patient's helmet, worn during his 3-hour daily motorcycle rides, which placed repeated tension on the periauricular area. This rare case illustrates how physical force contributes to auricular and periauricular keloid development and progression. It also shows that when performing surgery with periauricular incisions, care should be taken to eliminate wound/scar stretching.

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.

Low-grade Cribriform Cystadenocarcinoma: A Review of the Literature and Case Report.

Low-grade cribriform cystadenocarcinoma (LGCCC) is a rare tumor of the salivary gland that most often arises from the parotid gland. A 51-year-old man developed a small mass on the right parotid gland 5 years ago. A preoperative magnetic resonance image showed abnormal intensity, an atypical characteristic for such a tumor; therefore, the diagnosis was difficult. Thus, a superficial parotidectomy was performed as a total excisional biopsy to remove the tumor. Histopathological analyses revealed that the tumor was composed of a single cyst comprising cells containing mucosal fluid, with proliferation of large cells. Also, proliferation of the tumor epithelium showed a papillary cribriform pattern of proliferation with a partial ring form, and the tissue inside the tumor was replaced by a hematoma. Mild cellular atypia was observed. Immunostaining for S-100 was positive, and the Ki-67 ratio was <5%. These histopathological findings led to a diagnosis of LGCCC of the parotid gland. At 54 months after surgery, the patient has had no recurrence or facial palsy. LGCCC is a rare neoplasm of the salivary gland and is listed in the current World Health Organization classification (2005) as a variant of cystadenocarcinoma. This case suggests that a thorough preoperative examination can lead to better diagnosis of rare tumors, including LGCCC. Thus, if a plastic surgeon is to correctly diagnose and treat parotid grand tumors, including LGCCC, then a detailed preoperative examination, including imaging, a disease course review, a physical examination, and differential diagnosis, should be considered carefully.

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.