A Tri-fusion Reporter Mouse Reveals Tissue-Specific FGF1B Promoter Activity in vivo.

Transgenic mice harboring imaging reporters take full advantage of imaging technologies in studies using living mice. Here, we established a tri-fusion multimodal reporter gene containing fragments from firefly luciferase, enhanced green fluorescent protein, and herpes simplex virus type 1 thymidine kinase and generated tri-fusion reporter Tg mice. Fibroblast growth factor type 1 (FGF1), a multifunctional mitogen to a wide range of tissues, regulates proliferation of neural stem cells of the brain, where FGF1 expression is initiated through activation of the FGF1B (F1B) promoter. The reporter mouse under the control of the human F1B promoter enables visualization in vivo where F1B activity is elevated, including tissues not only in the brain but also in the nasopharynx, skull, spine, and testes, particularly in Leydig cells. Treating Tg mice with the alkylating agent busulfan, which is known to eradicate Leydig cells and disrupt spermatogenesis in mice, eliminated the reporter signals. Restoring Leydig cells recovered reporter expression, indicating that the reporter can be used as a surrogate marker for Leydig cells. The F1B tri-fusion reporter mouse model can be utilized in longitudinal monitoring of the health status of the male reproductive system, such as in studies exploring the toxicity of chemicals to spermatogenesis.

RU486-inducible recombination in the salivary glands of lactoferrin promoter-driven green fluorescent Cre transgenic mice.

When compared with the many tamoxifen-activated Cre mouse lines available for gene manipulation studies, relatively few RU486-inducible Cre mice are in use, due to leakiness issues. Here, we report the generation of an RU486-inducible triple fusion gene (GCrePR1e), consisting of green fluorescent protein, Cre, and the progesterone receptor ligand-binding domain (F642-L901). We sought to improve the GCrePR1e by selecting a truncated human lactoferrin (Lf) promoter to drive its expression, based on the promoter's low basal activity and innate sensitivity to RU486. The resulting vector displayed decreased leakiness and increased Cre induction by RU486 through transcriptional and posttranslational regulation in in vitro transfection assays. Inducible GCrePR1e expression was found in most organs of Lf-GCrePR1e transgenic mice and highly activated in the salivary gland, spleen, and lymph nodes. In the bigenic mouse generated by crossing the Lf-GCrePR1e mouse and the Cre reporter mouse (R26R-LacZ), we found that RU486-induced LacZ expression only in the mucous acini and striated ducts of the salivary gland and had very low background recombination in the untreated mice. Our results demonstrated that the Lf-CrePR1e vector was suitable for in vitro recombination in culture models, and Lf-CrePR1e transgenic mice could mediate spatially restricted and RU486-induced gene manipulation in the salivary gland.

Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells.

BACKGROUND: Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs) of young (8-10 weeks), adult (5 months), and old (21 months) mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. RESULTS: We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h) increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. CONCLUSIONS: We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases.

The use of injectable spherically symmetric cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation.

Typical cell transplantation techniques involve the administration of dissociated cells directly injected into muscular tissues; however, retention of the transplanted cells at the sites of the cell graft is frequently limited. An approach, using spherically symmetric aggregates of cells with a relatively uniform size self-assembled in a thermo-responsive methylcellulose hydrogel system, is reported in the study. The obtained cell aggregates preserved their endogenous extracellular matrices (ECM) and intercellular junctions because no proteolytic enzyme was used when harvesting the cell aggregates. Most of the cells within aggregates (with a radius of approximately 100 microm) were viable as indicated by the live/dead staining assay. After injection through a needle, the cell aggregates remained intact and the cells retained their activity upon transferring to another growth surface. The cell aggregates obtained under sterile conditions were transplanted into the skeletal muscle of rats via local injection. The dissociated cells were used as a control. It was found that the cell aggregates can provide an adequate physical size to entrap into the muscular interstices and offer a favorable ECM environment to enhance retention of the transplanted cells at the sites of the cell graft. These results indicated that the spherically symmetric cell aggregates developed in the study may serve as a cell delivery vehicle for therapeutic applications.

Multi-ion-crosslinked nanoparticles with pH-responsive characteristics for oral delivery of protein drugs.

pH-Responsive nanoparticles composed of chitosan (CS) and poly-gamma-glutamic acid (gamma-PGA) blended with tripolyphosphate (TPP) and MgSO(4) (multi-ion-crosslinked NPs) were prepared and characterized to determine their effectiveness in the oral delivery of insulin. Their counterparts without TPP and MgSO(4) (NPs) were used as a control. FT-IR and XRD results indicated that the spontaneous interaction between CS, insulin, gamma-PGA, MgSO(4) and TPP can form an ionically crosslinked network-structure, leading to the formation of nanoparticles. Multi-ion-crosslinked NPs were more compact than NPs, while their zeta potential values were comparable. During storage, multi-ion-crosslinked NPs suspended in deionized water were stable for at least 10 weeks. Multi-ion-crosslinked NPs had a superior stability over a broader pH range than NPs. In the in vitro release study, NPs failed to provide an adequate retention of loaded insulin in dissolution media compared to multi-ion-crosslinked NPs. Transepithelial-electrical-resistance and transport experiments demonstrated that multi-ion-crosslinked NPs significantly more effectively transported insulin than NPs; confocal visualization further validated the enhanced permeation of insulin via the paracellular pathway. The aforementioned results suggest that multi-ion-crosslinked NPs are a promising carrier for improved transmucosal delivery of insulin in the small intestine.

Human breast tumor cells express multimodal imaging reporter genes.

OBJECTIVE: Human ZR75-1 cells were among the first few characterized estrogen-dependent mammary gland carcinoma cell lines and had been utilized in various studies for the pro- or antitumor effect of xenoestrogens and antiestrogens. The objective of this study was to establish a breast tumor model in ZR75-1 cells bearing multimodal reporter genes to allow noninvasive imaging of tumor growth using fluorescence and nuclear imaging platforms. METHODS AND RESULTS: Enhanced green fluorescent protein (eGFP) cDNA was fused at the C-terminus with herpes simplex virus type 1 thymidine kinase (HSV1-tk) to form the fusion reporter gene (eGFP-tk). In vitro proliferation, migration, and invasion assays revealed that eGFP-tk-transfected ZR75-1 cells exhibited decreased proliferation rate, migratory activity, and invasion ability compared to the wild-type cells. The functional HSV1-tk enzymatic activity in stably transfected cells were confirmed by in vitro ganciclovir (GCV) sensitivity and [123I]2-fluoro-2-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU) accumulation assays. In vivo fluorescence and nuclear imaging were performed on nude mice bearing multiple subcutaneous xenografts established from ZR75-1-eGFP-tk and wild-type cells. Optical imaging was able to detect the green fluorescence of eGFP-tk tumor. The eGFP-tk reporter gene-specific imaging was achieved by single photon emission computed tomography (SPECT) using [123I]FIAU as a radiotracer and demonstrated decreased FIAU uptake in eGFP-tk tumor by GCV treatment. Probably due to a flare reaction after GCV treatment, micro-positron emission tomography (micro-PET) imaging using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) could not demonstrate decreases in FDG uptake. However, in vitro metabolic assay also revealed that eGFP-tk cells transiently increased [3H]-deoxyglucose uptake in response to GCV treatment. CONCLUSIONS: This study confirmed the usefulness of eGFP-tk in many applications by providing, in vitro and in vivo, the sensitive and reporter gene-specific imaging. ZR75-1-eGFP-tk cells that are ready to incorporate in various imaging platforms constitute a useful model in breast cancer research.

Stability of angiogenic agents, ginsenoside Rg1 and Re, isolated from Panax ginseng: in vitro and in vivo studies.

The study was designed to investigate the stability of ginsenoside Rg(1) (Rg(1)) and Re (Re), two natural herbal compounds isolated from Panax ginseng, based on their activity to promote angiogenesis in vitro and in vivo. After being treated at different temperatures, pHs, and solvent species for distinct durations, the remaining activities of Rg(1) and Re on human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation were examined in vitro. Additionally, the remaining activity of each treated test agent, mixed in a growth factor-reduced Matrigel, in stimulating angiogenesis was evaluated subcutaneously in a mouse model. Basic fibroblast growth factor (bFGF) was used as a control. It was found in vitro that HUVEC proliferation, migration in a Transwell plate, and tube formation on Matrigel were all significantly enhanced in the presence of bFGF, Rg(1), or Re. However, after being treated at different temperatures, pHs, or solvent species, the remaining activity of bFGF on HUVEC behaviors reduced significantly. This observation was more significant with increasing the duration of treatment. In contrast, the activities of Rg(1) and Re remained unchanged throughout the entire course of the study. The in vivo results observed on day 7 after implantation showed that the blank control (Matrigel alone) was slightly vascularized. In contrast, the density of neo-vessels in the Matrigel plug mixed with bFGF, Rg(1), or Re was significantly enhanced. However, after being treated, the density of neo-vessels was significantly reduced in the Matrigel plug mixed with bFGF, while those of Rg(1) and Re remained unchanged. The aforementioned results suggested that Rg(1) and Re could be a novel group of nonpeptide angiogenic agents with a superior stability and may be used for the management of tissue regeneration.

Shear stress regulates gene expression in vascular endothelial cells in response to tumor necrosis factor-alpha: a study of the transcription profile with complementary DNA microarray.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Myocyte protection by 10 kD heat shock protein (Hsp10) involves the mobile loop and attenuation of the Ras GTP-ase pathway.

Heat shock proteins (hsp), hsp60 and hsp10, are involved in the folding of imported mitochondrial proteins and the refolding of denatured proteins after stress. We examined whether hsp10 can reduce myocyte death by its mitochondrial function or by interacting with cytoplasmic signaling pathways. Overexpression of hsp10 by adenoviral infection decreased myocyte death induced by hydrogen peroxide, sodium cyanide, and simulated ischemia and reoxygenation (SI/RO). We generated an adenoviral vector coding for a temperature-sensitive mutant hsp10 protein (P34H), incapable of cooperatively refolding denatured malate dehydrogenase with hsp60. Overexpression of the hsp10 mutant potentiated SI/RO-induced myocyte death. Analysis of electron transport chain function revealed increased Complex I capacity with hsp10 overexpression, whereas hsp10(P34H) overexpression decreased Complex II capacity. Hsp10 overexpression preserved both Complex I and II function after SI/RO. Examination of the Ras GTP-ase signaling pathway indicated that inhibition of Ras was required for protection by hsp10. Constitutive activation of Ras abolished the effects afforded by hsp10 and hsp10(P34H). Hsp10 overexpression inactivated Raf, ERK, and p90Ribosomal kinase (p90RSK) before and after SI/RO. Our results suggest that complex mechanisms are involved in the protection by hsp10 against SI/RO-induced myocyte death. This mechanism may involve the hsp10 mobile loop and attenuation of the Ras GTP-ase signaling pathway.

Overexpression of PHGPx and HSP60/10 protects against ischemia/reoxygenation injury.

Reactive oxygen species arising from ischemia/reperfusion (I/R) cause damage to cardiac tissue. We examined the effects of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) and cytosolic PHGPx (cPHGPx) overexpression on protection against simulated I/R in neonatal rat cardiac myocytes (NCM). Additionally, a protective combinatorial effect with heat shock proteins 60 and 10 (HSP60/10) was investigated. NCM were infected with adenoviral vectors expressing mPHGPx, cPHGPx, HSP60/10, or an empty control (Adv-) and submitted to 8 h of ischemia followed by 16 h of reoxygenation. mPHGPx infection led to a 40% decrease in malondialdehyde and 4-hydroxy-2(E)-nonenal following I/R (p<.05). Creatine kinase and lactate dehydrogenase release were decreased in both mPHGPx-infected and HSP60/10-infected cells (p<.05). The combination of mPHGPx and HSP60/10 overexpression led to further protection (p<.01). DNA laddering and histone-associated DNA fragments were decreased in PHGPx- and HSP60/10-infected cells (p<.01). Cytochrome c release from mitochondria was decreased in mPHGPx-infected cells. Furthermore, mPHGPx overexpression preserved electron transport chain complex IV function following simulated I/R (p<.05). These results indicate that overexpression of PHGPx provides protection against damage resulting from simulated I/R injury, particularly in the mitochondria, and that the combination of mPHGPx and HSP60/10 imparts an added protective effect.