Won Bae Jeon

Young capillary vessels rejuvenate aged pancreatic islets

Significance The regulation of blood glucose is a homeostatic process that declines with age, but it is unknown whether this disturbance is a consequence of intrinsic dysfunction of the regulatory organ, the pancreatic islet. In marked contrast to the widely held notion that the insulin-producing pancreatic beta cell loses function with wear and tear, and thus causes age-related disturbances in glucose homeostasis, we show that mouse and human beta cells are fully functional at advanced age. The pancreatic islet as an organ, however, is threatened by vascular senescence. Replacing the islet vasculature in aged islet grafts rejuvenates the islet and fully restores glucose homeostasis, indicating that islet blood vessels should be targeted to mitigate frail glucose homeostasis associated with aging. Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.

Alisol-B, a novel phyto-steroid, suppresses the RANKL-induced osteoclast formation and prevents bone loss in mice

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1alpha,25(OH)(2)D(3)-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10(-8)M 1alpha,25(OH)(2)D(3) caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), an analog of 1alpha,25(OH)(2)D(3), administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

Stimulation of fibroblasts and neuroblasts on a biomimetic extracellular matrix consisting of tandem repeats of the elastic VGVPG domain and RGD motif

Elastin-like proteins (ELPs) modeled after tropoelastin are favored in the development of biomimetic matrices due to their biocompatibility and the possibility to precisely control their environmental responsiveness, mechanical properties, and fate within the cells by recombinant DNA technology-mediated design at the gene level. However, a basic prerequisite in the use of ELPs as cell culture matrices is the presence of a biofunctionality that can induce adhesion-mediated signaling pathways. To activate fibronectin-integrin signaling events from a cell-matrix interface and direct cell survival and proliferation, we biosynthesized a modular ELP, represented as TGPG[VGRGD(VGVPG)₆]₂₀ WPC, consisting of alternating elastic (VGVPG)₆structural domains and cell-binding VGRGD motifs that are intended to emulate various aspects of extracellular matrix proteins. The inverse transition curves of [VGRGD(VGVPG)₆]₂₀ and (VGVPG)₁₄₀ overlapped with each other, indicating that one VGRGD sequence fused with six elastic pentapeptides did not disturb the thermal sensitivity of [VGRGD(VGVPG)₆]₂₀. The cell adhesion activity of [VGRGD(VGVPG)₆]₂₀ toward HEK293 fibroblasts and N2A neuroblasts was similar to that of native fibronectin. Upon contact with [VGRGD(VGVPG)₆]₂₀, the fibroblasts exhibited a flattened polygonal morphology, and the neuroblasts synthesized new DNA and proliferated. On the basis of these physiological changes, we concluded that RGD-functionalized ELP triggers the activation of signaling cascades within cells and can be used as an elastin-like matrix for mammalian cell culture.

Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

Detection of C-reactive protein on a functional poly(thiophene) self-assembled monolayer using surface plasmon resonance

The preparation of a new poly(thiophene) with pendant N-hydroxysuccinimide ester groups and its application to immobilization of biomolecules are reported. A thiophene derivative of N-hydroxysuccinimide ester was polymerized with FeCl3 in chloroform and the resulting poly(thiophene) was characterized by nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). This polymer reacts with amine-bearing molecules to yield new poly(thiophene) derivatives and the specific interactions at the side groups could be detected. Thus, a self-assembled monolayer (SAM) using the polymer was formed on a gold-coated quartz cell and anti-C-reactive protein (anti-CRP) was immobilized. The binding behavior of CRP on the surface was monitored by use of a surface plasmon resonance (SPR) sensor system.

Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides

BackgroundIntegrin-mediated interaction of neuronal cells with extracellular matrix (ECM) is important for the control of cell adhesion, morphology, motility, and differentiation in both in vitro and in vivo systems. Arg-Gly-Asp (RGD) sequence is one of the most potent integrin-binding ligand found in many native ECM proteins. An elastin-mimetic recombinant protein, TGPG[VGRGD(VGVPG)6]20WPC, referred to as [RGD-V6]20, contains multiple RGD motifs to bind cell-surface integrins. This study aimed to investigate how surface-adsorbed recombinant protein can be used to modulate the behaviors and differentiation of neuronal cells in vitro. For this purpose, biomimetic ECM surfaces were prepared by isothermal adsorption of [RGD-V6]20 onto the tissue culture polystyrene (TCPS), and the effects of protein-coated surfaces on neuronal cell adhesion, spreading, migration, and differentiation were quantitatively measured using N2a neuroblastoma cells.ResultsThe [RGD-V6]20 was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V6]20 or fibronectin followed hyperbolic binding kinetics saturating around 2 μM protein concentration. The apparent maximum cell binding to [RGD-V6]20 was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V6]20 and fibronectin occurring at a coating concentration of 2.4 × 10-7 and 1.4 × 10-7 M, respectively. The percentage of spreading cells was in the following order of proteins: fibronectin (84.3% ± 6.9%) > [RGD-V6]20 (42.9% ± 6.5%) > [V7]20 (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V6]20 was similar to that of cells on fibronectin. The expression of neuronal marker proteins Tuj1, MAP2, and GFAP was approximately 1.5-fold up-regulated by [RGD-V6]20 relative to TCPS. Moreover, by the presence of both [RGD-V6]20 and RA, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated.ConclusionWe have shown that an elastin-mimetic protein consisting of alternating tropoelastin structural domains and cell-binding RGD motifs is able to stimulate neuronal cell behaviors and differentiation. In particular, adhesion-induced neural differentiation is highly desirable for neural development and nerve repair. In this context, our data emphasize that the combination of biomimetically engineered recombinant protein and isothermal adsorption approach allows for the facile preparation of bioactive matrix or coating for neural tissue regeneration.

Improved neural progenitor cell proliferation and differentiation on poly(lactide‐co‐glycolide) scaffolds coated with elastin‐like polypeptide

Poly(lactide-co-glycolide) (PLGA) and elastin-like polypeptide (ELP) have been widely used as a biodegradable scaffold and thermoresponsive matrix, respectively. However, little attention has focused on the combinatorial use of these biomaterials for tissue engineering applications. An ELP matrix TGPG[VGRGD(VGVPG)6]20WPC (referred to as REP) contains multiple Arg-Gly-Asp motifs. This study fabricated porous PLGA scaffolds coated with various concentration of matrix via thermally induced phase transition to improve adhesion-mediated proliferation and differentiation of neural progenitor cells. Matrix-coated scaffolds were characterized by FTIR, SEM, and hematoxylin and eosin staining with respect to coating efficiency, porosity, and pore size and shape. On the matrix-coated scaffolds, cells grew as a single cell or associated each other to form a multicellular layer or cluster. In biological evaluations, cell adhesion and proliferation were significantly promoted in a matrix concentration-dependent manner. More importantly, in combination with retinoic acid, the differentiation of progenitor cells into neuronal and astroglial lineages was highly stimulated in the cells cultured on matrix-coated scaffolds than on untreated controls. Taken together, our results indicated that the REP matrix-functionalized PLGA scaffolds are suitable for improving neuronal cell functions, and thus applicable for neural tissue engineering.

Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions

In this study, we aimed to compare the morphogenetic and neuronal characteristics between monolayer cells and spheroids. For this purpose, we established spheroid formation by growing SH-SY5Y cells on the hydrophobic surfaces of thermally-collapsed elastin-like polypeptide. After 4 days of culture, the relative proliferation of the cells within spheroids was approximately 92% of the values for monolayer cultures. As measured by quantitative assays for mRNA and protein expressions, the production of synaptophysin and neuronspecific enolase (NSE) as well as the contents of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins are much higher in spheroids than in monolayer cells. Under the all-trans-retinoic acid (RA)-induced differentiation condition, spheroids extended neurites and further up-regulated the expression of synaptophysin, NSE, CAMs, and ECM proteins. Our data indicate that RA-differentiated SH-SY5Y neurospheroids are functionally matured neuronal architectures. [BMB Reports 2013; 46(5): 276-281]

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair.

A new selective ‘turn-on’ small fluorescent cationic probe for recognition of RNA in cells

Fluorescent imaging probes have revolutionised cell biology by monitoring cellular objects. However, the lack of fluorescent probes with high selectivity for RNA has been a drawback. Thus, selective RNA binding for fluorescent sensors is essential. Here, we report the selective fluorescence enhancement upon addition of RNA. By exploiting a selective recognition of small tetra-cationic probe 1 for RNA, we also explain the possible binding mode for RNA. As a membrane-permeant fluorescence probe, 1 provides selective imaging of RNA not only in human neuroblastoma tumour SH-SY5Y cell line used for Parkinsons disease but also in the unicellular green alga cells. Further exploitation could open new opportunities in neurotoxin and cancer biology.