Young-Il Yang

Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-γ-induced upregulation of B7-H1 (CD274)

Majority of cancer cells upregulate co‐inhibitory molecule B7‐H1 which confers resistance to anti‐tumor immunity, allowing cancers to escape from host immune surveillance. We addressed the molecular mechanism underlying the regulation of cancer‐associated B7‐H1 expression in response to interferon‐γ (IFN‐γ). Using promoter constructs in luciferase assay, the region between 202 and 320 bp from the translational start site is responsible for B7‐H1 expression. Electrophoretic mobility shift assay, site‐directed mutagenesis and knockdown experiment using siRNA revealed that interferon regulatory factor‐1 (IRF‐1) is primarily responsible for the constitutive B7‐H1 expression as well as for the IFN‐γ‐mediated B7‐H1 upregulation in a human lung cancer cell line A549. Additionally, AG490, a Janus activated kinase/signal transducer and activator of transcription inhibitor, greatly abolished the responsiveness of A549 cells to IFN‐γ by reducing the IRF‐1 transcription. Our findings support a critical role of IRF‐1 in the regulation of constitutive and IFN‐γ‐induced expression of B7‐H1 in cancer cells.

The -238 tumor necrosis factor-α promoter polymorphism is associated with decreased susceptibility to cancers

We investigated the potential association of tumor necrosis factor-alpha (TNF-alpha) promoter polymorphisms with cancers. The study included 169 patients with gastric cancer, uterine cervical cancer, colorectal cancer, or renal cell carcinoma and 92 healthy controls. The -308 and -238 polymorphisms in the TNF-alpha promoter were analyzed by PCR-restriction fragment length polymorphism (RFLP). The proportion of individuals carrying the TNF-238A allele was significantly lower in the cancer group than in the control group. The odds ratio for cancer in subjects with the TNF-238A allele was 0.25 (95% CI, 0.10-0.64). No association was found between the -308 polymorphism and cancers. These results suggest that the -238A allele has a protective function against cancers.

Controlled release of cell-permeable gene complex from poly(L-lactide) scaffold for enhanced stem cell tissue engineering.

The use of tissue engineering to deliver genes to stem cells has been impeded by low transfection efficiency of the inserted gene and poor retention at the target site. Herein, we describe the use of non-viral gene transfer by cell-permeable peptide (CPP) to increase the transfection efficiency. The combination of this technique with the use of a controlled release concept using a poly (l-lactide) scaffold allowed for prolonged uptake in stem cells. High transfection efficiency was obtained using a human-derived arginine-rich peptide denoted as Hph-1 (YARVRRRGPRR). The formation of complex between pDNA and Hph-1 was monitored using gel retardation tests to measure size and zeta potential. Complex formation was further assessed using a DNase I protection assay. A sustained gene delivery system was developed using a fibrous 3-D scaffold coated with pDNA/Hph-1 complexes. Transfection efficiency and the mean fluorescence intensity of human adipose-derived stem cells (hASCs) on the sustained delivery scaffold were compared to those of cells transfected via bolus delivery. Plasmid DNA completely bound Hph-1 at a negative-to-positive (N/P) charge ratio of 10. After complex formation, Hph-1 appeared to effectively protect pDNA against DNase I attack and exhibited cytotoxicity markedly lower than that of the pDNA/PEI complex. Plasmid DNA/Hph-1 complexes were released from the scaffolds over 14days and were successfully transfected hASCs seeded on the scaffolds. Flow cytometry revealed that the transfection efficiency in hASCs treated with pDNA/Hph-1 complex was approximately 5-fold higher than that in cells transfected using Lipofectamine. The sustained delivery system showed a significantly higher transfection efficiency and remained able to transfect cells for a longer period of time than bolus delivery. These results suggest that cell-scaffold-based tissue regeneration can be further improved by transduction concept using CPP and controlled release using polymeric scaffold.

NFI-C regulates osteoblast differentiation via control of osterix expression.

In bone marrow, bone marrow stromal cells (BMSCs) have the capacity to differentiate into osteoblasts and adipocytes. Age‐related osteoporosis is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow. In this study, we demonstrate that disruption of nuclear factor I‐C (NFI‐C) impairs osteoblast differentiation and bone formation, and increases bone marrow adipocytes. Interestingly, NFI‐C controls postnatal bone formation but does not influence prenatal bone development. We also found decreased NFI‐C expression in osteogenic cells from human osteoporotic patients. Notably, transplantation of Nfic‐overexpressing BMSCs stimulates osteoblast differentiation and new bone formation, but inhibits adipocyte differentiation by suppressing peroxisome proliferator‐activated receptor gamma expression in Nfic−/− mice showing an age‐related osteoporosis‐like phenotype. Finally, NFI‐C directly regulates Osterix expression but acts downstream of the bone morphogenetic protein‐2‐Runx2 pathway. These results suggest that NFI‐C acts as a transcriptional switch in cell fate determination between osteoblast and adipocyte differentiation in BMSCs. Therefore, regulation of NFI‐C expression in BMSCs could be a novel therapeutic approach for treating age‐related osteoporosis. Stem Cells 2014;32:2467–2479

The biological activities of (1,3)-(1,6)-β-d-glucan and porous electrospun PLGA membranes containing β-glucan in human dermal fibroblasts and adipose tissue-derived stem cells

In this study, we investigated the possible roles of (1,3)-(1,6)-beta-d-glucan (beta-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing beta-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that beta-glucan and porous PLGA membranes containing beta-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with beta-glucan. Furthermore, we confirmed that porous PLGA membranes containing beta-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing beta-glucan interacted favorably with the membrane and the topical administration of beta-glucan was useful in promoting wound healing. Therefore, our study suggests that beta-glucan and porous PLGA membranes containing beta-glucan may be useful as a material for enhancing wound healing.

Epicardial delivery of VEGF and cardiac stem cells guided by 3-dimensional PLLA mat enhancing cardiac regeneration and angiogenesis in acute myocardial infarction

Congestive heart failure is mostly resulted in a consequence of the limited myocardial regeneration capacity after acute myocardial infarction. Targeted delivery of proangiogenic factors and/or stem cells to the ischemic myocardium is a promising strategy for enhancing their local and sustained therapeutic effects. Herein, we designed an epicardial delivery system of vascular endothelial growth factor (VEGF) and cardiac stem cells (CSCs) using poly(l-lactic acid) (PLLA) mat applied to the acutely infarcted myocardium. The fibrous VEGF-loaded PLLA mat was fabricated by an electrospinning method using PLLA solution emulsified VEGF. This mat not only allowed for sustained release of VEGF for 4weeks but boosted migration and proliferation of both endothelial cells and CSCs in vitro. Furthermore, sustained release of VEGF showed a positive effect on in vitro capillary-like network formation of endothelial cells compared with bolus treatment of VEGF. PLLA mat provided a permissive 3-dimensional (3D) substratum that led to spontaneous cardiomyogenic differentiation of CSCs in vitro. Notably, sustained stimulation by VEGF-loaded PLLA mat resulted in a substantial increase in the expression of proangiogenic mRNAs of CSCs in vitro. The epicardially implanted VEGF-loaded PLLA mat showed modest effects on angiogenesis and cardiomyogenesis in the acutely infarcted hearts. However, co-implantation of VEGF and CSCs using the PLLA mat showed meaningful therapeutic effects on angiogenesis and cardiomyogenesis compared with controls, leading to reduced cardiac remodeling and enhanced global cardiac function. Collectively, the PLLA mat allowed a smart cargo that enabled the sustained release of VEGF and the delivery of CSCs, thereby synergistically inducing angiogenesis and cardiomyogenesis in acute myocardial infarction.

Interleukin-4 C-590T polymorphism is associated with protection against nasal polyps in a Korean population.

Background Although many studies have implicated interleukin (IL)-4 promoter polymorphisms as potential determinants of disease susceptibility, there are no reports on the association between IL-4 promoter polymorphisms and nasal polyps. The aim of this study was to investigate the relationship between an IL-4 promoter polymorphism and nasal polyps. Methods The C-590T promoter polymorphism of the IL-4 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 106 Korean chronic rhinosinusitis patients with or without nasal polyps and 70 healthy Korean subjects. Results The frequency of the T allele at position -590 of the IL-4 gene in a Korean population was 0.85, which was significantly higher than those of other ethnic groups, and the T/T allele at position -590 of IL-4 was associated with protection against nasal polyps if compared with the C/C allele (relative risk, 0.529; 95% confidence interval, 0.307–0.912; p = 0.028). Conclusion T-590, which is dominant at position -590 of the IL-4 promoter, appears to be associated with a protective mechanism against nasal polyps in Korean populations.

In vitro study of osteogenic differentiation of bone marrow stromal cells on heat-treated porcine trabecular bone blocks

This in vitro study investigated the potential of the heat-treated porcine trabecular bone block as a bone substitute for the treatment of bone defects or related diseases. Chemical, mechanical, and morphological studies of bone blocks were performed. The resultant properties were compared with the properties of currently available commercial products from bovine trabecular bones. The major component of the bone block was hydroxyapatite, and the ratio of Ca/P was 1.65-1.66. The average values of the compressive Youngs modulus and the ultimate strength were 346.33 +/- 83.15 and 6.66 +/- 1.62 MPa, respectively. The pore size of the heat-treated bone blocks was approximately 300-500 microm. For the biological investigations, expanded bone marrow stromal cells (BMSCs) were isolated from the femurs of New Zealand White rabbits and were dynamically seeded into the heat-treated porcine bone block (10x10x5 mm3). Before the cells were seeded, the heat-treated porcine bone blocks were divided into two groups: collagen coated blocks (n=16) and uncoated blocks (n=16). Within each group, the blocks were again divided into two groups, depending on the culture method, i.e., static or rotating culture. Cells were cultured in the blocks for up to 6 weeks. Scanning electron microscopic examination after 4 weeks showed that the cell layers attached to the porcine bone block. Proliferation and osteogenic differentiation were analyzed by cell counting, an MTT assay, alkaline phosphatase activity, and total protein content. The deposition of extracellular substances and osteoid formation surrounded by osteoblast-like cuboidal cells were confirmed through histochemical staining and transmission electron microscopy. Based on the results of this study, we conclude that heat-treated porcine trabecular bone has great potential as a bone substitute and may even be superior to currently available commercial products.

The isolation and in situ identification of MSCs residing in loose connective tissues using a niche-preserving organ culture system.

Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.

Expression of pro-angiogenic cytokines and their inhibition by dexamethasone in an ex vivo model of nasal polyps.

We used an organ culture of nasal polyps (NP) to provide ex vivo model to study the expression of pro-angiogenic cytokines and the effect of glucocorticoids in the pathogenesis of NP. Glucocorticoids are the drugs of choice for clinical treatment of NP; however, their mechanism of action is not fully understood. The cell-cell and cell-matrix integrity is well maintained in cultured NP. Expression of IL-6, IL-8, bFGF, GRO, and MCP-1 was increased in cultured NP compared to pre-cultured NP. Expression levels of IL-6, bFGF, and GRO in cultured NP were downregulated by dexamethasone (DEX) treatment, while MCP-1 expression was not suppressed. Further, RT-PCR and immunohistochemical analysis showed that HIF-1alpha and VEGF were suppressed in DEX-treated NP compared to untreated NP. Taken together, these results suggest that ex vivo organ culture can be considered a useful model to study the pathogenesis and regulation of pro-angiogenic cytokines in nasal polypogenesis.