PKC Regulates YAP Expression through Alternative Splicing of YAP 3'UTR Pre-mRNA by hnRNP F.

The Yes-associated protein (YAP) is a transcriptional co-activator that plays critical roles in organ development and tumorigenesis, and is verified to be inhibited by the Hippo signaling pathway. In the present study, we show that the YAP 3'UTR is alternatively spliced to generate a novel 950 bp 3'UTR mRNA from the full length 3'UTR region (3483 bp) in human cancer cells. The ratio of full length 3'UTR YAP mRNA to alternatively spliced 3'UTR YAP mRNA is up-regulated by exposure of the cells to PKC inhibitor chelerythrine chloride. Further study using luciferase reporter assay showed that the expression of the alternatively spliced 3'UTR mRNA is much lower compared with the full length 3'UTR mRNA, suggesting that alternatively spliced 3'UTR YAP mRNA may have a shorter half-life than full length 3'UTR mRNA. Interestingly, PKC represses YAP 3'UTR-mediated mRNA stability is dependent on a splicing factor, hnRNP F. Activation of PKC induces nuclear translocation of cytosolic hnRNP F. Ectopic expression of hnRNP F enhances YAP 3'UTR splicing. Our results suggest that hnRNP F regulates YAP 3'UTR-mediated mRNA stability in an alternative splicing-dependent manner, and PKC regulated YAP expression is dependent on nuclear translocation of hnRNP F in human cancer cell lines.

MicroRNA 630 Represses NANOG Expression through Transcriptional and Post-Transcriptional Regulation in Human Embryonal Carcinoma Cells.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Dietary Leucine Supplement Ameliorates Hepatic Steatosis and Diabetic Nephropathy in db/db Mice.

Dietary leucine supplementation has been explored for the therapeutic intervention of obesity and obesity-induced metabolic dysfunctions. In this study, we aim to examine the effects of dietary leucine supplementation in db/db mice. Mice were treated with or without leucine (1.5% w/v) in drinking water for 12 weeks. The leucine supplement was found to reduce insulin resistance and hepatic steatosis in db/db mice. Using Nuclear Magnetic Resonance (NMR)-based lipidomics, we found that the reduction of hepatic triglyceride synthesis was correlated with attenuated development of fatty liver. In addition, diabetic nephropathy (DN) was also ameliorated by leucine. Using liquid chromatography⁻time-of-flight mass spectrometry (LC-TOF MS)-based urine metabolomics analysis, we found that the disturbance of the tricarboxylic acid (TCA) cycle was reversed by leucine. The beneficial effects of leucine were probably due to AMP-activated protein kinase (AMPK) activation in the liver and kidneys of db/db mice. Thus, dietary leucine supplementation may potentially be a nutritional intervention to attenuate hepatic steatosis and early DN in type II diabetes.

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo.

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling. METHODS: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment. RESULTS: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx 2 were up-regulated, while GSK-3ß was down-regulated after HBO treatment. Western blot analysis showed an increased ß-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. CONCLUSIONS: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines.

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Transcriptional regulators of the ΔNp63: their role in limbal epithelial cell proliferation.

The surface cells of corneal epithelium are regularly shed off and replaced by new cells that are derived from limbal epithelial stem cells (LESC). LESC are believed to reside in the basal layer of the limbal epithelium and are characterized with high expression levels of ΔNp63, a transcription factor (TF) which is believed to play roles in the regulation of LESC proliferation. In this study, we examined the transcriptional regulation of ΔNp63 in limbal epithelial cell. We employed DNA pull down assay followed by LC/MS analysis and cDNA microarray analysis to identify the TFs that were capable of binding to ΔNp63 promoter or were expressed at higher levels in limbus over cornea. The TFs thus selected were further examined for their in vivo ΔNp63 promoter binding by chromatin immunoprecipitation assay. We identified six putative TFs (PAX6, EGR1, CEBPB, JUN, ATF3, and ARID5B) through the aforementioned approaches. Among them, PAX6 and EGR1 were shown to promote the transcription of ΔNp63 and led to increased cell proliferation. In contrast, CEBPB and ATF3 appeared to exert little or no effect on ΔNp63 expression, however, their silencing suppressed cell proliferation. Although JUN exhibited low promoter-binding specificity, however, it affected ΔNp63 expression and limbal epithelial cell proliferation in ways similar to that of PAX6 and EGR1. Intriguingly, ARID5B was highly expressed in the limbal epithelial cell, however, its silencing by siRNA did not obviously affect the expression of ΔNp63, nor did it reduce cell proliferation of the limbal epithelial cell.

Preclinical experiments on the release behavior of biodegradable nanofibrous multipharmaceutical membranes in a model of four-wall intrabony defect.

Guided tissue regeneration (GTR) therapy has been widely used to regenerate lost periodontium from periodontal disease. However, in terms of regenerative periodontal therapy, a multidrug-loaded biodegradable carrier can be even more promising in dealing with periodontal disease. In the current study, we fabricated biodegradable nanofibrous collagen membranes that were loaded with amoxicillin, metronidazole, and lidocaine by an electrospinning technique. The in vitro release behavior and the cytotoxicity of the membranes were investigated. A four-wall intrabony defect was created in rabbits for in vivo release analysis. The bioactivity of the released antibiotics was also examined. The experimental results showed that the drug-loaded collagen membranes could provide sustainable release of effective amoxicillin, metronidazole, and lidocaine for 28, 56, and 8 days, respectively, in vivo. Furthermore, the bioactivity of the released antibiotics remained high, with average bioactivities of 50.5% for amoxicillin against Staphylococcus aureus and 58.6% for metronidazole against Escherichia coli. The biodegradable nanofibrous multipharmaceutical membranes developed in this study may provide a promising solution for regenerative periodontal therapy.

Neurodegeneration in streptozotocin-induced diabetic rats is attenuated by treatment with resveratrol.

AIM: Diabetes mellitus-associated hyperglycemia and oxidative stress have been shown to have detrimental effects on the brain and may lead to impairment of cognitive functions. Resveratrol (Rsv), a polyphenolic antioxidant, has been shown to have moderate hypoglycemic and prominent hypolipidemic effects in diabetic rats. In the present study, we examined if Rsv improves the diabetic encephalopathy and explored its possible underlying mechanisms. METHODS: Male SD rats were treated with streptozotocin (65 mg/kg), and the diabetic rats were orally fed with Rsv (0.75 mg/kg, every 8 h) or normal saline for 4 weeks. Animals were then sacrificed and the brain tissues (hippocampus) processed for biochemical and histological studies. RESULTS: Neurodegeneration and astrocytic activation were noted in the hippocampus of the diabetic rats. Tumor necrosis factor-α, IL-6 transcripts and nuclear factor-κB expression were increased in the brain. In addition, neuropathic alterations in the hippocampus were evident in diabetic rats, including increased blood vessel permeability and VEGF expression, decreased mitochondrial number and AMP-activated protein kinase activity. In Rsv-treated diabetic rats, the aforementioned abnormalities were all attenuated. CONCLUSION: These observations suggest that Rsv significantly attenuated neurodegeneration and astrocytic activation in the hippocampus of diabetic rats. Our results suggested that Rsv could potentially be a new therapeutic agent for diabetic encephalopathy and neurodegeneration.

Up-regulation of heat shock protein 70-1 (Hsp70-1) in human limbo-corneal epithelial cells cultivated on amniotic membrane: A proteomic study.

Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.

Resveratrol ameliorates metabolic disorders and muscle wasting in streptozotocin-induced diabetic rats.

Diabetes mellitus (DM) is characterized by dysregulated energy metabolism. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in diabetic animals. However, its overall in vivo effects on energy metabolism and the underlying mechanism require further investigation. In the present study, electrospray ionization-tandem mass spectrometry was employed to characterize the urine and plasma metabolomes of control, streptozotocin-induced DM and RSV-treated DM rats. Using principal component analysis (PCA) and heat map analysis, we discovered significant differences among control and experimental groups. RSV treatment significantly reduced the metabolic abnormalities in DM rats. Compared with the age-matched control rats, the level of carnitine was lower, and the levels of acetylcarnitine and butyrylcarnitine were higher in the urine and plasma of DM rats. RSV treatment ameliorated the deranged carnitine metabolism in DM rats. In addition, RSV treatment attenuated the diabetic ketoacidosis and muscle protein degradation, as evidenced from the attenuation of elevated urinary methyl-histidine and plasma branched-chain amino acids levels in DM rats. The beneficial effects of RSV in DM rats were correlated with activation of hepatic AMP-activated protein kinase and SIRT1 expression, increase of hepatic and muscular mitochondrial biogenesis and inhibition of muscle NF-κB activities. We concluded that RSV possesses multiple beneficial metabolic effects in insulin-deficient DM rats, particularly in improving energy metabolism and reducing protein wasting.

The growth-promoting effect of KGF on limbal epithelial cells is mediated by upregulation of DeltaNp63alpha through the p38 pathway.

Corneal epithelial stem cells are thought to reside in the limbus, the transition zoon between cornea and conjunctiva. Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are two paracrine factors that regulate the proliferation, migration and differentiation of the limbal epithelial cells; however, the underlying mechanisms are still poorly understood. In an ex vivo limbal explant culture, we found that KGF is a more potent growth stimulator for the epithelial outgrowth than HGF. Immunofluorescence studies of the epithelial outgrowth from cells treated with HGF or KGF showed similar expression patterns of keratin-3 and keratin-14. Interestingly, p63 was highly expressed in KGF-treated limbal epithelial sheets but not in those treated with HGF. Kinase inhibitor studies showed that induction of DeltaNp63alpha expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous DeltaNp63alpha was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of DeltaNp63alpha. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with DeltaNp63alpha expression as one of the downstream targets.

Resveratrol enhances perforin expression and NK cell cytotoxicity through NKG2D-dependent pathways.

In a previous report, we showed that the in vivo cytotoxic activity of the natural killer (NK) cells isolated from resveratrol-pretreated rats is significantly enhanced compared with that of the non-pretreated rats; however, the underlying mechanism remains unclear. In the present study, we use cultured NK92 cell line to examine the possible signaling pathways underlying the resveratrol-induced activation. Using cultured K562, HepG2, and A549 cells as targets, we show that resveratrol pretreatment increases NK cell cytotoxicity in a dose-dependent manner. The enhanced cytotoxic effect is accompanied by increases in JNK and ERK-1/2 MAP kinase activity and perforin expression. Moreover, the expression of NKG2D, an upstream signaling molecule of the MAP kinases pathway, is also enhanced. Resveratrol-enhanced perforin expression and cytotoxic activity are effectively inhibited by pretreatment with the inhibitors of JNK (SP600125), ERK-1/2 (PD98059), or by siRNAs against JNK-1 and ERK-2. However, the inhibitors or siRNA to p38 exhibits no effect. Since IL-2 has been shown to induce NKG2D expression and perforin release, we therefore, examined whether IL-2 and resveratrol act in parallel. We show that IL-2 also stimulates perforin expression, however, when treated together with resveratrol, they exhibit no additive effect. The results suggest that in NK92 cells, resveratrol may act via a similar or overlapping pathway as that of IL-2, to enhance perforin expression and cytotoxic activity. Data presented strongly indicate that resveratrol act via NKG2D-dependent JNK and ERK-1/2 pathways.

Transcriptional activity of TAp63 promoter is regulated by c-jun.

The transcription factor p63 belongs to the p53 protein family and plays an important role in epithelial development. Recent studies showed that p63 is over-expressed in some human squamous cell carcinomas of the head and neck, suggesting a role in carcinogenesis. The p63 gene contains two promoters and alternative promoter usage generates two groups of proteins with (TAp63) or without (ΔNp63) the transactivation domain. Although the roles of TAp63 in epithelial development have been described in numerous recent studies, the regulation of its expression has not been elucidated. In this study, we showed that the transcriptional activity of the TAp63 promoter and TAp63 protein level were both up-regulated by an increased c-jun activity in Hep3B human hepatocellular carcinoma cell. Moreover, the elevated TAp63 expression was coincided with an increased binding of c-jun to the TAp63 promoter. Point mutation of the sp1 binding site within the TAp63 promoter region attenuated the effect of c-jun on TAp63 expression. Knockdown of TAp63 expression by shRNA led to increased proliferation of Hep3B cell compared to that of the mock cell, suggesting a growth suppressive effect of TAp63.

TAp63 plays compensatory roles in p53-deficient cancer cells under genotoxic stress.

p53, p63, and p73 belong to the p53 family of proteins, which mediate development, differentiation, and various other cellular responses. p53 is involved in many anti-cancer mechanisms, such as cell cycle regulation, apoptosis, and the maintenance of genomic integrity. The p63 gene is controlled by two promoters that direct the expression of two isoforms, one with and one without transactivating properties, known as TAp63 and ΔNp63. In this study, p53-deficient cells (Hep3B and PC-3) and p53-expressing cells (A549 and HepG2) were treated with doxorubicin to examine the possible roles of TAp63 in these cells under genotoxic stress; TAp63 expression was induced in p53-deficient cell lines, but not in p53-expressing cell lines. The ectopic expression of p53 in p53-deficient cells (Hep3B) reduced TAp63 promoter activity, and knockdown of TAp63 attenuated doxorubicin-induced cell growth arrest by promoting cell cycle progression, leading to an increase in the percentage of G(2)/M cells. Moreover, knockdown of TAp63 increased cell sensitivity to doxorubicin-induced genomic damage. Our results suggest that TAp63 may play a compensatory role in cell cycle regulation and DNA damage repair in p53-deficient cancer cells.

Resveratrol ameliorates vasculopathy in STZ-induced diabetic rats: role of AGE-RAGE signalling.

BACKGROUND: Resveratrol (RSV) has been shown to ameliorate hyperglycaemia and hyperlipidaemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial effects of RSV on diabetes mellitus (DM)-induced vasculopathy and explored its possible mechanism. METHODS: Male Sprague-Dawley rats were injected with streptozotocin at 65 mg/kg body weight The induction of DM was confirmed by a fasting plasma glucose level > or = 300 mg/dL and symptoms of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75 mg/kg body weight three times a day for 4-8 weeks. Animals were sacrificed and vessel wall histology was examined by microscopy. The vascular smooth muscle cell activation was assessed by the medial thickness, collagen deposition, and the expressions receptor for advanced glycation end product, NF-kappaB, proliferation cell nuclear antigen, and the levels of Erk1/2 phosphorylation. RESULTS: In RSV-treated DM rats, the vascular wall thickening, collagen deposition/cross-linking, and vascular permeability were all alleviated compared with that of the untreated DM rats. The vascular smooth muscle cell of the RSV-treated rats was characterized with less proliferation, lower NF-kappaB, and Erk1/2 activation, decreased proliferation cell nuclear antigen and receptor for advanced glycation end product expression. Moreover, the plasma fructosamine was significantly reduced in RSV-treated DM rats. CONCLUSIONS: RSV alleviated DM-induced vasculopathy through attenuation of advanced glycation end product-receptor for advanced glycation end product-NF-kappaB signalling pathway.

Overexpression of delta Np63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation.

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The DeltaNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. DeltaNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of DeltaNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a DeltaNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of DeltaNp63 in cell proliferation and cell-cycle regulation. We found that the proliferation of NPC-076 cell is greatly suppressed when the overexpressed DeltaNp63 is silenced by specific DeltaNp63 siRNA. Further studies show that DeltaNp63 silencing results in the upregulation of CKIs, including p27(kip1) and p57(kip2) in both mRNA and protein levels. Cell-cycle analysis shows that DeltaNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that DeltaNp63 plays important roles in the regulation of NPC-076 cell-cycle progression, and may play a role in the maintenance of NPC-076 tumor cell phenotype.

Resveratrol ameliorates Serratia marcescens-induced acute pneumonia in rats.

Serratia marcescens is an important nosocomial pathogen, which has been especially problematic as a cause of hospital-acquired pneumonia in the past two decades. Treatment of S. marcescens-related infections has been limited by emergence of multiple drug-resistant strains. Thus, the development of alternative agents for the prevention and treatment of Serratia infection is urgently needed. Resveratrol (RSV) is a compound with diverse biological effects including anti-cancer, anti-inflammation, anti-diabetes, and cancer chemoprevention. Whether RSV has in vivo prophylactic or therapeutic potential against infection remains uncharacterized. In the present study, we used a murine acute pneumonia model initiated by intratracheal application of S. marcescens to evaluate whether RSV possesses anti-infection properties. We showed that pretreatment with RSV for 3 days markedly increased alveolar macrophage infiltration, elevated NK cell activity, and decreased bacterial burden in the infected lung with a subsequent decrease in mortality. These effects were associated with significantly less-severe inflammatory phenotypes in lung tissue and bronchoalveolar lavage fluid, including reduced neutrophil infiltration of the lungs, reduced phagocytosis activity, and reduced secretion of cytokines such as TNF-alpha, IL-1beta, and IL-6. To further characterize the underlying mechanism responsible for these effects of RSV, LPS derived from S. marcescens was used to induce acute pneumonia in rats, with or without RSV pretreatment. RSV was shown to ameliorate acute pneumonia via inhibition of the NF-kappaB signaling pathway, including inhibition of IkappaBalpha phosphorylation and subsequent NF-kappaB activation. These findings suggest that RSV might be beneficial as a prophylactic treatment in patients at risk of an episode of S. marcescens-induced acute pneumonia.

Exercise affects platelet-promoted tumor cell adhesion and invasion to endothelium.

This investigation explored how exercise intensity impacts platelet-mediated interactions of nasopharyngeal carcinoma cells (NPCs) and vascular endothelial cells (ECs) under shear flow in 33 males. Our results showed that (a) platelet-NPC aggregates (PNA) were associated with higher shear-induced P-selectin expression and glycoprotein alpha(II)beta(3) activation than platelet-platelet aggregates (PPA); (b) strenuous exercise (SE, up to VO2max), but not moderate exercise (ME, 60% VO2max for 30 min), increased both PPA and PNA in mimicked venous and arterial circuits and enhanced PNA in mimicked flow of stenotic vessels; (c) the percentages of PNA that remained bound to ECs in mimicked flow of post-capillary venules increased, while platelet-induced CD44 cleavage on NPC and trans-endothelial migration of NPC were enhanced following SE, but were unchanged in response to ME. We conclude that SE, but not ME, enhances the capacity of PNA to adhere to ECs, withstand flowing blood, and facilitate the invasion of NPCs toward ECs.

Matrix revolution: molecular mechanism for inflammatory corneal neovascularization and restoration of corneal avascularity by epithelial stem cell transplantation.

Corneal neovascularization (CNV) associated with severe limbal stem cell (LSC) deficiency remains a challenging ocular surface disease in that corneal inflammation may persist and progress, and the condition will not improve without LSC transplantation. A prominent feature after successful LSC transplantation is the suppression of corneal inflammation and CNV, which is generally attributed to the endogenous anti-angiogenic/anti-inflammatory factors secreted by corneal epithelial cells. In addition, corneal epithelial basement membrane (EBM) plays a unique role in the regulation of angiogenesis; several potent anti-angiogenic factors are derived from the matrix component of EBM, such as endostatin (from collagen XVIII) and restin (from collagen XV). Also, angio-inhibitory thrombospondin and tissue inhibitor of metalloproteinase-3 are deposited in EBM. Moreover, the heparan sulphate proteoglycan in EBM can bind and sequester VEGF and FGF-2 from activation. Recently, cultivated corneal epithelial transplantation (CCET) and cultivated oral mucosal epithelial transplantation (COMET) have emerged as promising techniques for the treatment of LSC deficiency. When human limbo-corneal epithelial (HLE) cells are cultivated on cryopreserved amniotic membrane, production of endostatin, restin, and IL-1ra is enhanced. This highlights the significance of delicate epithelial-matrix interactions in the generation of anti-angiogenic/anti-inflammatory factors by HLE cells, and this may, in part, explain the rapid restoration of corneal avascularity following CCET. In addition, whether epithelial stem cells can persist after transplantation is the key for CCET and COMET. Emerging evidence of long-term survival of cultivated epithelial cells after transplantation suggest that epithelial stem cells can be isolated and cultivated in vitro, and can re-establish the epithelial phenotype in vivo. Taken together, the merits of enhanced anti-angiogenic activity and the preservation of corneal epithelial stem cells encourage further application of this tissue engineering technique for ocular surface reconstruction.