Bromodeoxyuridine Labeling Research Articles

The effectiveness of epigallocathechin-3-gallate for treatment of human papillomavirus-driven epithelial neoplasms: a preclinical study

BackgroundEpigallocathechin-3-gallate (EGCG), the major bioactive polyphenol in green tea, has anticarcinogenic properties in vitro and in vivo. Several recent reports have shown that EGCG affects the expression of human papillomavirus (HPV)-encoded E6 and E7, two oncoproteins required for HPV-driven oncogenesis. Here, we aimed to explore the effects of EGCG on keratinocyte proliferation and differentiation, in addition to HPV18 replication, in a three-dimensional organotypic raft culture system. MethodsOrganotypic raft cultures of HPV18-infected keratinocytes cultured at the air–liquid interface for 10 days were treated with EGCG for an additional 10 days before fixation and processing. Raft sections were stained with antibodies specific for various cell proliferation and keratinocyte differentiation markers in addition to tumour suppressor genes. Western blotting was performed on EGCG-treated cells to measure the level of HPV18 E6 and E7 protein expression. FindingsEGCG treatment blocked the ability of HPV18-positive keratinocytes to generate hyperplastic epithelium in raft culture. EGCG reduced cell proliferation as assessed by bromodeoxyuridine label incorporation and Ki67 staining; it upregulated expression of several tumour suppressor genes (p53 [TP53], p21, pRb), and impaired productive viral replication (as assessed by HPV18 E4 protein expression), but did not have an effect on keratinocyte differentiation. In culture, EGCG treatment promoted degradation of the E6 and E7 proteins and restored tumour suppressor gene expression. InterpretationThe results of our preclinical study suggest that EGCG inhibits the proliferation of HPV18-infected keratinocytes by enhancing the turnover and degradation of the E6 and E7 proteins, resulting in re-expression of several key tumour suppressor genes. These findings suggest that EGCG could potentially reverse the dysplastic changes induced by oncogenic HPV strains and could be used clinically to treat HPV-induced neoplasia. FundingCancer Research UK.

Candesartan Restores the Amyloid Beta-Inhibited Proliferation of Neural Stem Cells by Activating the Phosphatidylinositol 3-Kinase Pathway.

Background and PurposeNeurogenesis in the adult brain is important for memory and learning, and the alterations in neural stem cells (NSCs) may be an important aspect of Alzheimer's disease (AD) pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to have an important role in neuronal cell survival and is highly involved in adult neurogenesis. Candesartan is an angiotensin II receptor antagonist used for the treatment of hypertension and several studies have reported that it also has some neuroprotective effects. We investigated whether candesartan could restore the amyloid-β(25–35) (Aβ25-35) oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway.MethodsTo evaluate the effects of candesartan on the Aβ25-35 oligomer-inhibited proliferation of NSCs, the NSCs were treated with several concentrations of candesartan and/or Aβ25-35 oligomers, and MTT assay and trypan blue staining were performed. To evaluate the effect of candesartan on the Aβ-inhibited proliferation of NSCs, we performed a bromodeoxyuridine (BrdU) labeling assay. The levels of p85α PI3K, phosphorylated Akt (pAkt) (Ser473), phosphorylated glycogen sinthase kinase-3β (pGSK-3β) (Ser9), and heat shock transcription factor-1 (HSTF-1) were analyzed by Western blotting.ResultsThe BrdU assays demonstrated that NSC proliferation decreased with Aβ25-35 oligomer treatment; however, a combined treatment with candesartan restored it. Western blotting displayed that candesartan treatment increased the expression levels of p85α PI3K, pAkt (Ser473), pGSK-3β (Ser9), and HSTF. The NSCs were pretreated with a PI3K inhibitor, LY294002; the effects of candesartan on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked.ConclusionsTogether, these results suggest that candesartan restores the Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway.

Effect of combined VEGF165/ SDF-1 gene therapy on vascular remodeling and blood perfusion in cerebral ischemia.

OBJECTIVE Therapeutic neovascularization is a promising strategy for treating patients after an ischemic stroke; however, single-factor therapy has limitations. Stromal cell-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) proteins synergistically promote angiogenesis. In this study, the authors assessed the effect of combined gene therapy with VEGF165 and SDF-1 in a rat model of cerebral infarction. METHODS An adenoviral vector expressing VEGF165 and SDF-1 connected via an internal ribosome entry site was constructed (Ad- VEGF165-SDF-1). A rat model of middle cerebral artery occlusion (MCAO) was established; either Ad- VEGF165-SDF-1 or control adenovirus Ad- LacZ was stereotactically microinjected into the lateral ventricle of 80 rats 24 hours after MCAO. Coexpression and distribution of VEGF165 and SDF-1 were examined by reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence. The neurological severity score of each rat was measured on Days 3, 7, 14, 21, and 28 after MCAO. Angiogenesis and vascular remodeling were evaluated via bromodeoxyuridine and CD34 immunofluorescence labeling. Relative cerebral infarction volumes were determined by T2-weighted MRI and triphenyltetrazolium chloride staining. Cerebral blood flow, relative cerebral blood volume, and relative mean transmit time were assessed using perfusion-weighted MRI. RESULTS The Ad- VEGF165-SDF-1 vector mediated coexpression of VEGF165 and SDF-1 in multiple sites around the ischemic core, including the cortex, corpus striatum, and hippocampal granular layer. Coexpression of VEGF165 and SDF-1 improved neural function, reduced cerebral infarction volume, increased microvascular density and promoted angiogenesis in the ischemic penumbra, and improved cerebral blood flow and perfusion. CONCLUSIONS Combined VEGF165 and SDF-1 gene therapy represents a potential strategy for improving vascular remodeling and recovery of neural function after cerebral infarction.

Abstract 4071: Targeted disruption of T-cell protein tyrosine phosphatase in mouse epidermis reveals its critical role in chemically-induced skin carcinogenesis

Abstract Environmental factors such as chemical toxicants contribute to the development of skin cancer by creating mutations in housekeeping genes and proto-oncogenes which disrupt intracellular signaling mechanisms. One vital signaling mechanism is tyrosine phosphorylation signaling. Phosphotyrosine signaling is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Recently we reported TC-PTP (PTPN2) is critical regulator of STAT3 signaling in mouse keratinocytes. To assess the role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TC-PTP-deficient (K14Cre.PTPN2fl/fl) transgenic mice. Loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency in epidermis resulted in a significant increase in epidermal thickness and hyperproliferation (assessed by bromodeoxyuridine (BrdU) labeling index) compared to control mice following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Consistently, primary keratinocytes derived from TC-PTP-deficient mice showed a faster growth rate. To further investigate the role of TC-PTP in skin carcinogenesis, TC-PTP-deficient mice were subjected to two-stage skin carcinogenesis analysis. TC-PTP-deficient mice showed a shortened latency of tumor development and significantly increased numbers of tumors compared to control mice, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. The data reveals TC-PTP may be a novel potential target for the prevention of skin cancer due to its role in the regulation of proliferation and apoptosis in epidermis. Citation Format: Hyunseung Lee, Mihwa Kim, Liza D. Morales, Thomas J. Slaga, John DiGiovanni, Dae J. Kim. Targeted disruption of T-cell protein tyrosine phosphatase in mouse epidermis reveals its critical role in chemically-induced skin carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4071.

Serum translationally controlled tumor protein is involved in rat liver regeneration after hepatectomy.

The translationally controlled tumor protein (TCTP) has been reported to promote progression of many physiological processes. However, whether TCTP is involved in liver regeneration has been rarely studied. This study aimed to investigate the potential role of serum TCTP in liver regeneration after two-thirds partial hepatectomy. The synthesis rate and accumulated expression of TCTP was assessed by phosphor imaging and Western blot analysis, respectively. The mRNA expression of tctp was analyzed by quantitative real-time PCR. The effect of serum TCTP on hepatocyte proliferation was investigated by bromodeoxyuridine incorporation, liver/body weight ratio, albumin concentration, and histological examination of liver following treatment of rat with anti-TCTP antibody or prokaryotic TCTP protein before hepatectomy. The MTT assay was used to examine effect of TCTP on hepatocyte proliferation in vitro. The results showed that the expression of intracellular and serum TCTP protein was significantly increased in rats after two-thirds partial hepatectomy. In vivo bromodeoxyuridine labeling assay suggested that treatment with anti-TCTP antibody before hepatectomy significantly decreased hepatocyte proliferation and liver/body weight ratio. The prokaryotic TCTP had a potential promoting effect on hepatocyte proliferation both in vivo and in vitro, although prokaryotic TCTP given to rats prior to hepatectomy did not increase the proliferation ratio or liver/body weight ratio. Furthermore, anti-TCTP antibody pretreatment decreased the expression of cyclin E, cdk2, and interleukin-6 in rat liver. These findings suggest serum TCTP is involved in rat liver regeneration through promoting hepatocyte proliferation.

Hypoxia Inhibits De Novo Vascular Assembly of Adipose-Derived Stromal/Stem Cell Populations, but Promotes Growth of Preformed Vessels.

Vascularization is critical for cell survival within tissue-engineered grafts. Adipose-derived stromal/stem cells (ASCs) are widely used in tissue engineering applications as they are a clinically relevant source of stem cells and endothelial progenitor cells. ASCs have previously been shown to self-assemble into pericyte-stabilized vascular networks in normoxic (20% O2) cultures. This capacity for de novo vascular assembly may accelerate graft vascularization in vivo rather than relying solely on angiogenic ingrowth. However, oxygen depletion within large cell-seeded grafts will be rapid, and it is unclear how this worsening hypoxic environment will impact the vascular assembly of the transplanted cells. The objectives of this study were to determine whether ASC-derived vessels could grow in hypoxia and to assess whether the vessel maturity (i.e., individual cells vs. preformed vessels) influenced this hypoxic response. Utilizing an in vitro vascularization model, ASCs were encapsulated within fibrin gels and cultured in vitro for up to 6 days in either normoxia (20% O2) or hypoxia (0.2% or 2% O2). In a subsequent experiment, vessels were allowed to preform in normoxia for 6 days before an additional 6 days of either normoxia or hypoxia. Viability, vessel growth, pericyte coverage, proliferation, metabolism, and angiogenic factor expression were assessed for each experimental approach. Vessel growth was dramatically inhibited in both moderate and severe hypoxia (47% and 11% total vessel length vs. normoxia, respectively), despite maintaining high cell viability and upregulating endogenous expression of vascular endothelial growth factor in hypoxia. Bromodeoxyuridine labeling indicated significantly reduced proliferation of endothelial cells in hypoxia. In contrast, when vascular networks were allowed to preform for 6 days in normoxia, vessels not only survived but also continued to grow more in hypoxia than those maintained in normoxia. These findings demonstrate that vascular assembly and growth are tightly regulated by oxygen tension and may be differentially affected by hypoxic conditions based on the maturity of the vessels. Understanding this relationship is critical to developing effective approaches to engineer viable tissue-engineered grafts in vivo.

Transfection with thymidine kinase permits bromodeoxyuridine labelling of DNA replication in the human malaria parasite Plasmodium falciparum.

BackgroundPlasmodium falciparum, the causative agent of severe human malaria, is an early-diverging protozoan whose lifecycle has many unusual features, including its modes of replication. Research on the Plasmodium cell cycle, which occurs primarily via schizogony instead of canonical binary fission, has been hampered by a lack of tools and markers that can be transferred from cell cycle studies in model organisms. A common tool used to study DNA replication and the cell cycle in human cells is the labelling of newly-replicated DNA with the modified nucleotide bromodeoxyuridine (BrdU), followed by immunofluorescent detection. Plasmodium parasites, however, do not incorporate BrdU because they rely only on de novo synthesis of pyrimidines and do not salvage thymidine analogues like BrdU for conversion into nucleotides.MethodsAnalysis of biochemical pathways in Plasmodium indicated that the absence of the enzyme thymidine kinase (TK) may be the only impediment to BrdU incorporation in this organism. A TK gene from Herpes simplex was, therefore, introduced into the Plasmodium falciparum 3D7 strain and the effect on BrdU labelling was assessed by enzyme-linked immunosorbent assay and immunofluorescence microscopy.ResultsIntroduction of a TK gene produces parasites that can indeed incorporate BrdU. This forms a sensitive indicator of DNA replication, which can be detected by both quantitative and qualitative assays on either a population level or a single-cell level. Plasmodium falciparum, when expressing TK, becomes unusually sensitive to BrdU toxicity.ConclusionsBrdU labelling represents a significant new tool for investigating DNA replication and the cell cycle in Plasmodium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1014-7) contains supplementary material, which is available to authorized users.

Active populations of rare microbes in oceanic environments as revealed by bromodeoxyuridine incorporation and 454 tag sequencing

The “rare biosphere” consisting of thousands of low-abundance microbial taxa is important as a seed bank or a gene pool to maintain microbial functional redundancy and robustness of the ecosystem. Here we investigated contemporaneous growth of diverse microbial taxa including rare taxa and determined their variability in environmentally distinctive locations along a north–south transect in the Pacific Ocean in order to assess which taxa were actively growing and how environmental factors influenced bacterial community structures. A bromodeoxyuridine–labeling technique in combination with PCR amplicon pyrosequencing of 16S rRNA genes gave 215–793OTUs from 1200 to 3500 unique sequences in the total communities and 175–299OTUs nearly 860 to 1800 sequences in the active communities. Unexpectedly, many of the active OTUs were not detected in the total fractions. Among these active but rare OTUs, some taxa (2–4% of rare OTUs) showed much higher abundance (>0.10% of total reads) in the active fraction than in the total fraction, suggesting that their contribution to bacterial community productivity or growth was much larger than that expected from their standing stocks at each location. An ordination plot by the principal component analysis presented that bacterial community compositions among 4 sampling locations and between total and active fractions were distinctive with each other. A redundancy analysis revealed that the variability of community compositions significantly correlated to seawater temperature and dissolved oxygen concentration. Also, a variation partitioning analysis showed that the environmental factors explained 49% of the variability of community compositions and the distance only explained 4.0% of its variability. These results implied very dynamic change of community structures due to environmental filtering. The active bacterial populations are more diverse and spread further in rare biosphere than we have ever seen. This study implied that rare microbes are important as an active part of microbial communities functioning ecosystems.

Myocyte Dedifferentiation Drives Extraocular Muscle Regeneration in Adult Zebrafish.

The purpose of this study was to characterize the injury response of extraocular muscles (EOMs) in adult zebrafish. Adult zebrafish underwent lateral rectus (LR) muscle myectomy surgery to remove 50% of the muscle, followed by molecular and cellular characterization of the tissue response to the injury. Following myectomy, the LR muscle regenerated an anatomically correct and functional muscle within 7 to 10 days post injury (DPI). Following injury, the residual muscle stump was replaced by a mesenchymal cell population that lost cell polarity and expressed mesenchymal markers. Next, a robust proliferative burst repopulated the area of the regenerating muscle. Regenerating cells expressed myod, identifying them as myoblasts. However, both immunofluorescence and electron microscopy failed to identify classic Pax7-positive satellite cells in control or injured EOMs. Instead, some proliferating nuclei were noted to express mef2c at the very earliest point in the proliferative burst, suggesting myonuclear reprogramming and dedifferentiation. Bromodeoxyuridine (BrdU) labeling of regenerating cells followed by a second myectomy without repeat labeling resulted in a twice-regenerated muscle broadly populated by BrdU-labeled nuclei with minimal apparent dilution of the BrdU signal. A double-pulse experiment using BrdU and 5-ethynyl-2'-deoxyuridine (EdU) identified double-labeled nuclei, confirming the shared progenitor lineage. Rapid regeneration occurred despite a cell cycle length of 19.1 hours, whereas 72% of the regenerating muscle nuclei entered the cell cycle by 48 hours post injury (HPI). Dextran lineage tracing revealed that residual myocytes were responsible for muscle regeneration. EOM regeneration in adult zebrafish occurs by dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. A mechanistic understanding of myocyte reprogramming may facilitate novel approaches to the development of molecular tools for targeted therapeutic regeneration in skeletal muscle disorders and beyond.

LXA4 actions direct fibroblast function and wound closure

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Cell Junction Pathology of Neural Stem Cells Is Associated With Ventricular Zone Disruption, Hydrocephalus, and Abnormal Neurogenesis.

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling, immunocytochemistry, electron microscopy, and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones, loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats, the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless, they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition, thereby opening the avenue for stem cell therapy.

Altitudinal patterns of diversity and functional traits of metabolically active microorganisms in stream biofilms.

Resources structure ecological communities and potentially link biodiversity to energy flow. It is commonly believed that functional traits (generalists versus specialists) involved in the exploitation of resources depend on resource availability and environmental fluctuations. The longitudinal nature of stream ecosystems provides changing resources to stream biota with yet unknown effects on microbial functional traits and community structure. We investigated the impact of autochthonous (algal extract) and allochthonous (spruce extract) resources, as they change along alpine streams from above to below the treeline, on microbial diversity, community composition and functions of benthic biofilms. Combining bromodeoxyuridine labelling and 454 pyrosequencing, we showed that diversity was lower upstream than downstream of the treeline and that community composition changed along the altitudinal gradient. We also found that, especially for allochthonous resources, specialisation by biofilm bacteria increased along that same gradient. Our results suggest that in streams below the treeline biofilm diversity, specialisation and functioning are associated with increasing niche differentiation as potentially modulated by divers allochthonous and autochthonous constituents contributing to resources. These findings expand our current understanding on biofilm structure and function in alpine streams.

Low-Dose Sevoflurane Promotes Hippocampal Neurogenesis and Facilitates the Development of Dentate Gyrus-Dependent Learning in Neonatal Rats

Huge body of evidences demonstrated that volatile anesthetics affect the hippocampal neurogenesis and neurocognitive functions, and most of them showed impairment at anesthetic dose. Here, we investigated the effect of low dose (1.8%) sevoflurane on hippocampal neurogenesis and dentate gyrus-dependent learning. Neonatal rats at postnatal day 4 to 6 (P4–6) were treated with 1.8% sevoflurane for 6 hours. Neurogenesis was quantified by bromodeoxyuridine labeling and electrophysiology recording. Four and seven weeks after treatment, the Morris water maze and contextual-fear discrimination learning tests were performed to determine the influence on spatial learning and pattern separation. A 6-hour treatment with 1.8% sevoflurane promoted hippocampal neurogenesis and increased the survival of newborn cells and the proportion of immature granular cells in the dentate gyrus of neonatal rats. Sevoflurane-treated rats performed better during the training days of the Morris water maze test and in contextual-fear discrimination learning test. These results suggest that a subanesthetic dose of sevoflurane promotes hippocampal neurogenesis in neonatal rats and facilitates their performance in dentate gyrus-dependent learning tasks.

Differentiation of human umbilical cord mesenchymal stem cells into germ-like cells in mouse seminiferous tubules.

Our previous study demonstrated that human umbilical cord mesenchymal stem cells (HUMSCs) were capable of differentiation into germ cells in vitro. To assess this potential in vivo, HUMSCs were microinjected into the lumen of seminiferous tubules of immunocompetent mice, which were treated with busulfan to destroy endogenous spermatogenesis. Bromodeoxyuridine labeling studies demonstrated that HUMSCs survived in the tubule for at least 120 days, exhibited a round cell shape typical of proliferating or differentiating germ cells, migrated to the basement of the tubule, where proliferating spermatogonia reside and returned to the luminal compartment, where differentiating spermatids and spermatozoa reside. The migration pattern resembled that of germ cell development in vivo. Immunohistochemical and colocalization studies revealed that transplanted HUMSCs expressed the germ cell markers octamer-binding transcription factor 4, α6 integrin, C-kit and VASA, confirming the germ cell differentiation. In addition, it was observed that tubules transplanted with HUMSCs exhibited marked improvement in the histological features damaged by the chemotherapeutic busulfan, as judged by morphology and quantitative histology. Taken together, these data demonstrated the capacity of HUMSCs to form germ cells in the testes and to repair testicular tissue. These findings suggest a potential utility of HUMSCs to treat the infertility and testicular insufficiency caused by cancer therapeutics.

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

Thyroid hormone signaling controls hair follicle stem cell function

Observations in thyroid patients and experimental animals show that the skin is an important target for the thyroid hormones. We previously showed that deletion in mice of the thyroid hormone nuclear receptors TRα1 and TRβ (the main thyroid hormone-binding isoforms) results in impaired epidermal proliferation, hair growth, and wound healing. Stem cells located at the bulges of the hair follicles are responsible for hair cycling and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of the bulge stem cells could be responsible for this phenotype. Bulge cells show increased levels of epigenetic repressive marks, can retain bromodeoxyuridine labeling for a long time, and have colony-forming efficiency (CFE) in vitro. Here we demonstrate that mice lacking TRs do not have a decrease of the bulge stem cell population. Instead, they show an increase of label-retaining cells (LRCs) in the bulges and enhanced CFE in vitro. Reduced activation of stem cells leading to their accumulation in the bulges is indicated by a strongly reduced response to mobilization by 12-O-tetradecanolyphorbol-13-acetate. Altered function of the bulge stem cells is associated with aberrant activation of Smad signaling, leading to reduced nuclear accumulation of β-catenin, which is crucial for stem cell proliferation and mobilization. LRCs of TR-deficient mice also show increased levels of epigenetic repressive marks. We conclude that thyroid hormone signaling is an important determinant of the mobilization of stem cells out of their niche in the hair bulge. These findings correlate with skin defects observed in mice and alterations found in human thyroid disorders.

Determining c-Myb protein levels can isolate functional hematopoietic stem cell subtypes.

The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c-Myb in HSCs although remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-Myb(low) HSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myb(low) and c-Myb(high) HSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior.

Cartilage-specific ablation of XBP1 signaling in mouse results in a chondrodysplasia characterized by reduced chondrocyte proliferation and delayed cartilage maturation and mineralization

To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage. Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation. Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR. Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.

Protein kinase C inhibition rescues manic-like behaviors and hippocampal cell proliferation deficits in the sleep deprivation model of mania.

Background:Recent studies revealed that bipolar disorder may be associated with deficits of neuroplasticity. Additionally, accumulating evidence has implicated alterations of the intracellular signaling molecule protein kinase C (PKC) in mania.Methods:Using sleep deprivation (SD) as an animal model of mania, this study aimed to examine the possible relationship between PKC and neuroplasticity in mania. Rats were subjected to SD for 72h and tested behaviorally. In parallel, SD-induced changes in hippocampal cell proliferation were evaluated with bromodeoxyuridine (BrdU) labeling. We then examined the effects of the mood stabilizer lithium, the antipsychotic agent aripiprazole, and the PKC inhibitors chelerythrine and tamoxifen on both behavioral and cell proliferation impairments induced by SD. The antidepressant fluoxetine was used as a negative control.Results:We found that SD triggered the manic-like behaviors such as hyperlocomotion and increased sleep latency, and reduced hippocampal cell proliferation. These alterations were counteracted by an acute administration of lithium and aripiprazole but not of fluoxetine, and only a single administration of aripiprazole increased cell proliferation on its own. Importantly, SD rats exhibited increased levels of phosphorylated synaptosomal-associated protein 25 (SNAP-25) in the hippocampus and prefrontal cortex, suggesting PKC overactivity. Moreover, PKC inhibitors attenuated manic-like behaviors and rescued cell proliferation deficits induced by SD.Conclusions:Our findings confirm the relevance of SD as a model of mania, and provide evidence that antimanic agents are also able to prevent SD-induced decrease of hippocampal cell proliferation. Furthermore, they emphasize the therapeutic potential of PKC inhibitors, as revealed by their antimanic-like and pro-proliferative properties.

Mir-135a enhances cellular proliferation through post-transcriptionally regulating PHLPP2 and FOXO1 in human bladder cancer.

BackgroundBladder cancer is the most common malignancy in urinary system and the ninth most common malignancy in the world. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. We investigated the expression of miR-135a in bladder cancer and explored its bio-function during bladder cancer progression.MethodsThe expression of miR-135a in bladder cancer cells and tissues are performed by using Real-time PCR assay. Cell viability assay (MTT assay), colony formation assay, anchorage-independent growth ability assay and Bromodeoxyuridine labeling and immunofluorescence (BrdUrd) assay are used to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis is used to determine cell cycle progression. The expressions of p21, p27, CyclinD1, Ki67, PHLPP2 and FOXO1 are measured by Western blotting assay. Luciferase assay is used to confirm whether FOXO1 is the direct target of miR-135a.ResultsmiR-135a is upregulated in bladder cancer cells and tissues. Enforced expression of miR-135a promotes bladder cancer cells proliferation, whereas inhibition of miR-135a reverses the function. Furthermore, for the first time we demonstrated PHLPP2 and FOXO1 are direct targets of miR-135a and transcriptionally down-regulated by miR-135a. Suppression of PHLPP2 or FOXO1 by miR-135a, consisted with dysregulation of p21, p27, Cyclin D1 and Ki67, play important roles in bladder cancer progression.ConclusionOur study demonstrates that miR-135a promotes cell proliferation in bladder cancer by targeting PHLPP2 and FOXO1, and is performed as an onco-miR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0438-8) contains supplementary material, which is available to authorized users.