Takeshi Kawazoe

Adipose Tissue–Specific Regulation of Angiotensinogen in Obese Humans and Mice: Impact of Nutritional Status and Adipocyte Hypertrophy

BACKGROUND The adipose tissue renin-angiotensin system (RAS) has been implicated in the pathophysiology of obesity and dysfunction of adipose tissue. However, neither regulation of angiotensinogen (AGT) expression in adipose tissue nor secretion of adipose tissue-derived AGT has been fully elucidated in humans. METHODS Human subcutaneous abdominal adipose tissue (SAT) biopsies were performed for 46 subjects with a wide range of body mass index (BMI). Considering the mRNA level of AGT and indices of body fat mass, the amount of adipose tissue-derived AGT secretion (A-AGT-S) was estimated. Using a mouse model of obesity and weight reduction, plasma AGT levels were measured with a newly developed enzyme-linked immunosorbent assay (ELISA), and the contribution of A-AGT-S to plasma AGT levels was assessed. RESULTS A-AGT-S was substantially increased in obese humans and the value was correlated with the plasma AGT level in mice. A-AGT-S and plasma AGT were higher in obese mice, whereas lower in mice with weight reduction. However, the AGT mRNA levels in the liver, kidney, and aorta were not altered in the mouse models. In both humans and mice, the AGT mRNA levels in mature adipocytes (MAs) were comparable to those in stromal-vascular cells. Coulter Multisizer analyses revealed that AGT mRNA levels in the MAs were inversely correlated with the average size of mature adipocytes. CONCLUSIONS This study demonstrates that adipose tissue-derived AGT is substantially augmented in obese humans, which may contribute considerably to elevated levels of circulating AGT. Adipose tissue-specific regulation of AGT provides a novel insight into the clinical implications of adipose tissue RAS in human obesity.

Effects of bFGF incorporated into a gelatin sheet on wound healing.

Basic fibroblast growth factor (bFGF) is well known to promote the proliferation of almost all cells associated with wound healing. However, as the activation duration of bFGF is very short in vivo, we incorporated bFGF into an acidic gelatin hydrogel and studied the sustained release of bFGF in vivo. In addition, we investigated the effects of the acidic gelatin sheet containing bFGF on wound healing. To distinguish wound contraction from neoepithelialization, we measured both the wound area and neoepithelium length. Other histological parameters such as thickness of granulation tissue and number of capillaries were also determined as indices of wound healing. Fibrous tissue was assessed using an Elastica van Gieson and Azan stain. A skin defect (1.5 × 1.5 cm) of full thickness was created on the back of each test mouse and the wound was covered with an acidic gelatin hydrogel, referred to as a gelatin sheet in this study (2 × 2 cm), with bFGF (100 μg/site) (A) or without bFGF (B). 1, 2, 3, 5, 7 and 14 days after covering, mice were killed and an enzyme-linked immunosorbent assay (ELISA) was performed to estimate the concentration of bFGF in the plasma. In another experiment, each wound was covered with (A), (B) or a hydrogel dressing (control group, C) and the wound area was measured 1 or 2 weeks postoperatively with a computer planimeter. The histological parameters, as mentioned above, were assessed using a light microscope. Sustained release of bFGF from the gelatin sheet was observed and the gelatin sheet containing bFGF promoted neoepithelialization, granulation, neovascularization and wound closure. This gelatin sheet containing bFGF was concluded to be effective for wound healing and promising for clinical use.

Enhanced wound healing by an epigallocatechin gallate-incorporated collagen sponge in diabetic mice

Epigallocatechin‐3‐O‐gallate (EGCG), the major polyphenolic compound present in green tea, has potent anti‐oxidant and free radical‐scavenging activities. In this study, various concentrations (10, 100, and 1,000 ppm) of EGCG were incorporated into a collagen sponge (CS) in order to investigate its healing effects on full‐thickness wounds created in type 2 diabetic mice. After 14 days, the residual wound size of the mice treated with 10 ppm EGCG‐incorporated collagen sponge (E‐CS) decreased significantly faster than that of the other mice. Moreover, significant increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of the capillaries were also histologically observed in the wound sites exposed to 10 ppm E‐CS in comparison with the others. Furthermore, 10 ppm E‐CS resulted in significant increases in the immunoreactivity of Ki‐67 (reepithelialization at the wound site), CD31 (formation of blood vessels), and α‐smooth muscle actin (the induction of myofibroblasts across the dermis). These results suggest that a CS incorporated with EGCG at low concentrations can enhance wound healing in diabetic mice by accelerating reepithelialization and angiogenesis as well as improving the cellular reorganization of granulation tissue by triggering the activity of myofibroblasts.

Tissue Augmentation by White Blood Cell-Containing Platelet-Rich Plasma:

Platelet-rich plasma (PRP) is a matrix of fibrin and platelets that releases cytokines that are important in wound healing. PRP is produced from the patients blood and therefore has less risk of allergic reaction and infection. We have obtained PRP with an enhanced white blood cell component (W-PRP) by optimizing the centrifugal separation of PRP from plasma. Here we show that injection of W-PRP into the auricle of nude mice gave greater tissue augmentation compared to PRP. Further augmentation occurred when bFGF was added to W-PRP, and there was a significant increase in the number of α-smooth muscle actin-positive cells in mice treated with W-PRP+bFGF. Our results suggest that W-PRP may have value in cosmetic surgery aimed at rejuvenation of wrinkled and sagging skin. W-PRP injection constitutes a new concept in cell transplantation, in which cells required for tissue regeneration are induced by cytokines released from the transplanted cells.

Incorporation of basic fibroblast growth factor into preconfluent cultured skin substitute to accelerate neovascularisation and skin reconstruction after transplantation

Cultured skin substitutes (CSS) with both epidermal and dermal components seem to be ideal, but they have not been widely used clinically, partly because it takes several weeks to produce them. Decreasing the number of seeding cells may reduce the period required for production, but it still takes a long time before the cells become confluent and neovascularisation is completed in CSS after grafting. As we have already succeeded in reducing the number of seeded keratinocytes in this study, we first attempted to reduce the number of seeded fibroblasts. Consequently, preconfluent CSS with 100×103cells/cm2 of fibroblasts combined with 100×103cells/cm2 of keratinocytes could be successfully grafted on to full-thickness wounds. bFGF-impregnated gelatin microspheres were then added to the preconfluent CSS before grafting. Incorporation of bFGF significantly accelerated neovascularisation and increased epidermal thickness, cellular components, and thickness of the dermis. The incorporation of bFGF makes CSS a potential therapeutic approach for management of skin wounds.

Reduction of progressive burn injury by using a new nonselective endothelin-A and endothelin-B receptor antagonist, TAK-044: an experimental study in rats.

&NA; Endothelins are well‐known vasoconstrictor peptides produced by vascular endothelial cells that have been reported to have a fundamental role in regulation of the systemic blood circulation. Plasma levels of endothelins are increased by burn injury, which also causes thrombosis and occlusion of vessels in the dermis as well as a vascular response in the adjacent uninjured dermis. Diminished blood flow leads to progressive ischemia and necrosis of the dermis beneath and around the burn (zone of stasis). If blood flow could be restored in this zone, secondary tissue damage would be minimized. In this study we examined the effects of a new nonselective endothelin receptor antagonist, TAK‐044 (Takeda Chemical Industries, Ltd., Osaka, Japan), on burn trauma in rats. Fifty male Sprague‐Dawley rats weighing an average of 450 gm were burned with a brass probe that produced a row of three burns 10 × 30 mm in size and two intervening unburned areas 5 × 30 mm in size. Rats were divided into five groups of 10 animals. Four groups received 0.01, 0.1, 1, or 10 mg/kg of TAK‐044 via the dorsal vein of the penis immediately after burn trauma, while the control group received the same volume of saline. Skin blood flow was measured with a laser‐Doppler flowmeter, and the development of edema and the area of necrotic tissue also were determined. Inhibition of endothelin activity by TAK‐044 after burn injury improved microvascular perfusion in the zone of stasis and prevented the progression of tissue damage in this zone. This supports the role of endothelins in the progression of burn injury in the zone of stasis. TAK‐044 was most effective in preventing progressive burn damage at a dose of 1 mg/kg. The extent of necrosis and edema was reduced significantly, and blood flow in the zone of stasis was increased in the treated rats.

Usefulness of palatal mucoperiosteal grafts for artificial eye socket contracture.

The authors performed palatal mucoperiosteal grafting for contracture of the artificial eye socket in 4 patients. Mucoperiosteal grafts were collected from the paramedian area of the hard palate. After release of contracture, the grafts were sutured with absorbable thread to the defective areas on the conjunctival side of the artificial eye socket after release of contracture. All patients showed mucoperiosteal graft survival without problems, no recurrence of contracture, and good courses of artificial eye wear. The mucoperiosteal donor areas showed closed healing after 3 to 4 weeks. Palatal mucoperiosteal grafts can be collected en bloc and are relatively rigid, which allows the simultaneous reconstruction of the conjunctival side and supportive tissue of the eyelid. Although the size of graft collection is limited, grafts with adequate size for partial reconstruction can be collected. Mucoperiosteal grafts are a good reconstruction material for contracture of the artificial eye socket.

Green tea polyphenols affect skin preservation in rats and improve the rate of skin grafts.

Green tea polyphenols have been recently reported to promote the preservation of tissues, such as blood vessels, corneas, nerves, islet cells, articular cartilage, and myocardium, at room temperature. These findings indicate the possibility of a new method of tissue banking without freezing. A main active ingredient of green tea, epigallocatechin-3-gallate (EGCG), is a polyphenol that possesses antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG regarding skin preservation. Skin sample biopsy specimens measuring 1 × 1 cm from GFP rats were held in sterile containers with 50 ml preserving solution at 4°C and 37°C for up to about 8 weeks. Periodically, some of the preserved skin specimens were directly examined histologically and others were transplanted into nude mice. Histological examinations of skin preserved at 4°C revealed a degeneration of the epidermal and dermal layers from 5 weeks in all groups. In the groups preserved at 37°C, degeneration and flakiness of the epidermal layer were demonstrated starting at 2 weeks preservation regardless of addition of EGCG. After 2–7 weeks of preservation the rat skin grafted to nude mice in the EGCG groups stored at 4°C showed successful engraftment. However, grafts preserved at 4°C without EGCG and at 37°C did not demonstrate GFP-positive keratinocyte or fibroblasts. In conclusion, the present findings suggest the future clinical usefulness of EGCG for skin preservation without freezing; however, the mechanism by which EGCG promotes skin preservation still remains unclear.

A retrospective analysis on the proper size of tissue expanders to treat scalp lesions.

Background: Tissue expanders have become established instruments for scalp reconstruction. However, selection of the size of the expander has not been investigated systematically, and it generally depends on the experience of the surgeon. Methods: We retrospectively analyzed 21 patients who had undergone treatment for scalp lesions using tissue expanders without any complications and measured 2 variables: the volume of the expanders per area of the excised lesions and the hypothetical stretched functional skin width relative to the width of the excised lesions. We also sought to evaluate the relationship between these 2 variables and the need for revision surgery during the postoperative course. Results: The need for revision surgery was statistically higher in patients with a volume of 7 ml/cm2 lesion or less and width of functional skin of less than 2.5 cm/cm lesion (P < 0.05). For scar repairs, the required size and volume of the expanders tended to be larger than those required for any other lesions. Conclusions: Expanders that generate functional skin at least more than 2.5 times the width of the lesion and have a volume more than 7 ml/cm2 lesion are necessary to cover scalp lesions without complications.

Long-term preservation of rat skin tissue by epigallocatechin-3-o-gallate.

Skin grafts can be preserved by cryopreservation and refrigerated storage at 4°C. Epigallocatechin-3-O-gallate (EGCG) enhances the viability of stored skin grafts and also extends the storage time up to 7 weeks at 4°C. EGCG, the major polyphenolic constituent present in green tea, has potent antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG on skin cryopreservation. Skin sample biopsy specimens from GFP rats were previously treated with/without EGCG then moved to −196°C. Skin samples were transplanted to nude mice after 2, 8, and 24 weeks of preservation. Glucose consumption was measured after thawing to assess the metabolic activity. Two weeks later the transplanted skin grafts were excised and histologically analyzed. Histological examinations revealed the degeneration of the epidermal and dermal layers in all groups. In the EGCG groups, the grafts showed higher integrity in the epidermal layer and dermal matrix. The present findings suggest the future clinical usefulness of EGCG for skin preservation; however, the mechanism by which EGCG promotes skin preservation still remains unclear.