Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined. Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated. ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia. ASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.

Background. The adipose tissue renin–angiotensin system (RAS) has been implicated in the pathophysiology of obesity and dysfunction of adipose tissue. H

Platelet-rich plasma (PRP) is an autologous blood product that contains a high concentration of several growth factors. Platelet-derived growth factor (PDGF)-BB is a potential mitogen for human adipose-derived stem cells (hASCs). PRP stimulates proliferation of hASCs; however, the signaling pathways activated by PRP remain unclear. hASCs were cultured with or without PRP or PDGF-BB, and proliferation was assessed. hASCs were also treated with PRP or PDGF-BB with or without imatinib, which is a PDGF receptor tyrosine kinase inhibitor, or sorafenib, which is a multikinase inhibitor. Inhibition of cell proliferation was examined using anti-PDGF antibody (Abcam, Cambridge, UK), by cell counting. We assessed the effects of inhibitors of various protein kinases such as ERK1/2, JNK, p38, and Akt on the proliferation of hASCs. The proliferation was remarkably promoted in cells treated with either 1% PRP or 10 ng/ml PDGF-BB, and both imatinib and sorafenib inhibited this proliferation. Anti-PDGF antibody (0.5 and 2 μg/ml) significantly decreased the proliferation of hASCs compared with control. PRP-mediated hASC proliferation was blocked by inhibitors of ERK1/2, Akt, and JNK, but not by an inhibitor of p38. PRP promotes hASC proliferation, and PDGF-BB in PRP plays a major role in inducing the proliferation of hASCs. PRP promotes hASC proliferation via ERK1/2, PI3K/Akt, and JNK signaling pathways.

Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in healthcare. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), which is capable of sustained release of basic fibroblast growth factor (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Patients with chronic skin ulcers that had not healed in at least 4 weeks were treated with CGS impregnated with bFGF at 7 or 14 μg/cm² after debridement, and the wound bed improvement was assessed 14 days after application. Wound bed improvement was defined as a granulated and epithelialized area on day 14 with a proportion to the baseline wound area after debridement of 50% or higher. The wound area, the wound area on day 14, and the granulation area on day 14 were independently measured by blinded reviewers in a central review using digital images of wounds taken with a calibrator. Patients were followed up until 28 days after application to observe the adverse reactions related to the application of CGS. From May 2010 to June 2011, 17 patients were enrolled and, in 16 patients, the wound bed improved. Among the randomized patients in step 2, no significant difference was seen between the low-dose group and the high-dose group. No serious adverse reactions were observed. Adverse reactions with a clear causal relationship to the study treatment were mild and patients quickly recovered from them. This study is the first-in-man clinical trial of CGS and showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. This combination therapy could be a promising therapy for chronic skin ulcers.