Transplantation of human cord blood-derived endothelial progenitor cells (EPCs) is reported to contribute to neovascularization in various ischemic diseases. However, the possible beneficial role and underlying mechanisms in diabetes-impaired wound healing have been less well characterized. In this study, EPC transplantation stimulated keratinocyte and fibroblast proliferation substantially as early as 3 days after injury, leading to significantly accelerated wound closure in streptozotocin-induced diabetic nude mice, compared to PBS control. RT-PCR analysis showed that EPCs secreted various wound healing-related growth factors. Among them, keratinocyte growth factor and platelet-derived growth factor were highly expressed in the EPCs and were present at substantial levels in the EPC-injected dermal tissue. Using EPC-conditioned medium (CM), we found that paracrine factors from EPCs directly exerted mitogenic and chemotactic effects on keratinocytes and fibroblasts. Moreover, injection of EPC-CM alone into the same diabetic wound mice promoted wound healing and increased neovascularization to a similar extent as achieved with EPC transplantation. These results indicate that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their direct paracrine action on keratinocytes, fibroblasts, and endothelial cells, rather than through their physical engraftment into host tissues (vasculogenesis). In addition, EPC-CM was shown to be therapeutically equivalent to EPCs, at least for the treatment of diabetic dermal wounds, suggesting that conditioned medium may serve as a novel therapeutic option that is free from allograft-associated immune rejection concern.

Objective-Although stem cell factor (SCF) has been shown to play a critical role in hematopoiesis, gametogenesis, and melanogenesis, the function of SCF in the regulation of vascular integrity has not been studied. Approach and Results-We demonstrated that SCF binds to and activates the cKit receptor in endothelial cells, thereby increasing the internalization of vascular endothelial-cadherin and enhancing extravasation of dyes to a similar extent as vascular endothelial growth factor. SCF-mediated cKit activation in endothelial cells enhanced the phosphorylation of endothelial nitric oxide (NO) synthase via the phosphoinositide 3-kinase/Akt signaling pathway and subsequently increased the production of NO. Inhibition of endothelial NO synthase expression and NO synthesis using small interfering RNA knockdown and chemical inhibitors substantially diminished the ability of SCF to increase the internalization of vascular endothelial-cadherin and in vitro endothelial permeability. SCF-induced increase in extravasation of the dyes was abrogated in endothelial NO synthase knockout mice, which indicates that endothelial NO synthase-mediated NO production was responsible for the SCF-induced vascular leakage. Furthermore, we demonstrated that the expression of SCF and cKit was significantly higher in the retina of streptozotocin-injected diabetic mice than in the nondiabetic control animals. Depletion of SCF by intravitreous injection of anti-SCF-neutralizing immunoglobulin G significantly prevented vascular hyperpermeability in the retinas of streptozotocin-injected diabetic mice. Conclusions-Our data reveal that SCF disrupts the endothelial adherens junction and enhances vascular leakage, as well as suggest that anti-SCF/cKit therapy may hold promise as a potential therapy for the treatment of hyperpermeable vascular diseases. previously described as a multifunctional cytokine involved in hematopoiesis, gametogenesis, and melanogenesis. [10][11][12] In divergent cell types, SCF has been demonstrated to play an essential role in the regulation of cell survival, proliferation, differentiation, and migration. However, the role of SCF in ECs has not yet been fully determined, although the results of several studies have shown that the SCF/cKit pathway contributes to angiogenesis and vascular regeneration. [13][14][15] Interestingly, the result of our previous studies revealed that SCF-mediated cKit activation in ECs and endothelial progenitor cells induced a phosphorylation of multiple downstream signaling molecules including eNOS.14 Given that eNOS activation and subsequent NO synthesis are key signaling events in eliciting endothelial hyperpermeability, we proposed that SCF might regulate endothelial permeability. In the present study, we aimed to investigate the novel role of SCF in the regulation of endothelial permeability, as well as to determine its contribution to retinal vascular leakage in streptozotocin (STZ)-injected diabetic mice. Materials and MethodsMaterials and Methods are available in the online-only Sup...

To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.

Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.