Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D₃ and β-glycerophosphate, respectively. In vivo, the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited β-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.     

Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro

Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.     

CUDC-101, a histone deacetylase inhibitor,improves the in vitro and in vivo developmental competence of somatic cell nuclear transfer pig embryos

The aim of the present study was to examine the effects of CUDC-101, a novel histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 at lysine 9 (AcH3K9) in pig SCNT embryos. We found that treatment with 1 μmol/L CUDC-101 for 24 hours significantly improved the development of pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.5% vs. 10.3%; P < 0.05). To assess in vivo developmental potency, CUDC-101–treated SCNT embryos were transferred into two surrogate mothers, resulting in one pregnancy with six fetuses. We then investigated the acetylation level of histone H3K9 in SCNT embryos treated with CUDC-101 and compared them only against untreated embryos. The acetylation level of control SCNT embryos was lower than that of CUDC-101–treated embryos at pseudo-pronuclear stages, and immunofluorescent signal for H3K9ac in CUDC-101–treated embryos in a pattern similar to that of control group. In conclusion, we demonstrated that CUDC-101 can significantly improve in vitro and in vivo developmental competence and enhance the nuclear reprogramming of pig SCNT embryos.     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

Engineering cell alignment in vitro

Cell alignment plays a critical role in various cell behaviors including cytoskeleton reorganization, membrane protein relocation, nucleus gene expression, and ECM remodeling. Cell alignment is also known to exert significant effects on tissue regeneration (e.g., neuron) and modulate mechanical properties of tissues including skeleton, cardiac muscle and tendon. Therefore, it is essential to engineer cell alignment in vitro for biomechanics, cell biology, tissue engineering and regenerative medicine applications. With advances in nano- and micro-scale technologies, a variety of approaches have been developed to engineer cell alignment in vitro, including mechanical loading, topographical patterning, and surface chemical treatment. In this review, we first present alignments of various cell types and their functionality in different tissues in vivo including muscle and nerve tissues. Then, we provide an overview of recent approaches for engineering cell alignment in vitro. Finally, concluding remarks and perspectives are addressed for future improvement of engineering cell alignment.     

Encapsulation reactions in vitro by haemocytes of Heliothis virescens

A simple in vitro system for studying capsule formation by Heliothis virescens haemocytes was devised. The system produced capsules morphologically and ultrastructurally similar to those formed in vivo. Encapsulation proceeded normally when melanization was inhibited and when divalent cations were absent. Capsule development took place in two physiologically distinct phases. Aggregation of haemocytes around a foreign object (phase 1) was a passive process. Consolidation of haemocytes into a smooth, adherent capsule (phase 2) required metabolic energy. Phase 1 was inhibited irreversibly by propranolol and caffeine. Inhibition of phase 1 by mild trypsinization could be reversed by haemolymph lysate. Preliminary evidence indicates that encapsulation promoting factors in the lysate originate in haemocytes.     

LYG-202, a new flavonoid with a piperazine substitution, shows antitumor effects in vivo and in vitro

LYG-202 is a newly synthesized flavonoid with a piperazine substitution. We investigated the antitumor effect of LYG-202 in vivo and in vitro. We show that, LYG-202 significantly decreases tumor growth in mice inoculated with S180 sarcoma cells, compared with the control group. Meanwhile, the viabilities of various kinds of tumor cells were inhibited by LYG-202 with IC₅₀ values in the range of 4.80 to 27.70 μM. Then apoptosis induced by LYG-202 in HepG2 cells was characterized by DAPI staining and Annexin V/PI double staining and degradation of PARP was observed. Activation of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9, and -3. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that LYG-202 exhibited strong antitumor effect in vivo and in vitro, involving with apoptosis induction.     

Ultra structure analysis of cell–cell interactions between pericytes and neutrophils in vitro

Neutrophils’ adhesion to the endothelium during inflammatory is a well-known processes. In contrast the interaction of neutrophils with cells of the neurovascular unit after they have been transmigrated into the brain is less clear. Recently, lymphocyte function-associated antigen-1 (LFA-1) dependent subendothelial crawling of neutrophils has been observed in vivo. This is mediated by intracellular adhesion molecule-1 (ICAM-1), which is expressed on the cell surface of pericytes. In our work we demonstrated in vitro a cell–cell interaction between porcine brain capillary pericytes (PBCPs) and neutrophils, with further characterization of the initial contact between these cells. PBCPs increase ICAM-1 protein expression in response to the cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, an increase in neutrophil adhesion to PBCPs was determined by immunofluorescence staining. By means of scanning force microscopy (SFM), we could additionally show that pericytes as well as neutrophils form cell extensions towards the neighboring cell. Interestingly, these extensions differ for different cell types.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [³H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

ErbB3 silencing reduces osteosarcoma cell proliferation and tumor growth in vivo

Osteosarcoma is the most common primary bone tumor in children and adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of the molecular signals that contribute to the aberrant osteosarcoma cell growth may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here we show that the expression of ErbB3 is increased in human osteosarcoma cells in vitro. Tissue microarray analysis of tissue cores from osteosarcoma patients further showed that the ErbB3 protein expression is higher in bone tumors compared to normal bone tissue, and is further increased in patients with recurrent disease or soft tissue metastasis. In murine osteosarcoma cells, silencing ErbB3 using shRNA decreased cell replication, cell migration and invasion, indicating that ErbB3 contributes to tumor cell growth and invasiveness. Furthermore, ErbB3 silencing markedly reduced tumor growth in a murine allograft model in vivo. Immunohistochemal analysis showed that the reduced tumor growth induced by ErbB3 silencing in this model resulted from decreased cell osteosarcoma cell proliferation, supporting a role of ErbB3 in bone tumor growth in vivo. Taken together, the results reveal that ErbB3 expression in human osteosarcoma correlates with tumor grade. Furthermore, silencing ErbB3 in a murine osteosarcoma model results in decreased cell growth and invasiveness in vitro, and reduced tumor growth in vivo, which supports the potential therapeutic interest of targeting ErbB3 in osteosarcoma.     

Brugia pahangi: Feeding and nutrient uptake in vitro and in vivo

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.