Kowit-Yu Chong

Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis

IntroductionIdiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.MethodsHypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.ResultsThe expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.ConclusionTransplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.

Lactoferrin as a natural regimen for selective decontamination of the digestive tract: Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonates from pathogenic challenge in the gastrointestinal tract

BACKGROUND Nosocomial infection with antibiotic-resistant strains is a major threat to critical care medicine. Selective decontamination of the digestive tract (SDD) is one of the strategies used to reduce ventilator-associated pneumonia and sepsis in critically ill patients. In the present study, we performed pathogenic challenges of the digestive tract in a transgenic milk-fed animal model to test whether porcine lactoferrin (pLF) is an effective SDD regimen. METHODS Transgenic mice expressing recombinant pLF in their milk at a mean+/-SD concentration of 120+/-13.6 mg/L during the lactation stage fed normal CD-1 mice pups for 4 weeks. The pups were subsequently challenged with pathogenic Escherichia coli, Staphylococcus aureus, and Candida albicans. RESULTS Compared with the control groups fed wild-type (normal) milk, the groups fed pLF-enriched milk demonstrated statistically significant improvements in weight gain; lower bacterial numbers in intestinal fluid, blood, and liver; healthier microvilli in the small intestine; and alveoli in the lungs. CONCLUSIONS Our results showed that oral administration of pLF-enriched milk to mice led to broad-spectrum antimicrobial activity in the digestive tract and protected the mucosa of the small intestine from injury, implying that pLF can be used as an effective SDD regimen.

Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis

Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.

cis-Resveratrol produces anti-inflammatory effects by inhibiting canonical and non-canonical inflammasomes in macrophages

Resveratrol, a natural phenolic compound found in red grapes and wine, exists as cis and trans isomers. Recent studies have shown that trans-resveratrol possesses anti-inflammatory, anti-oxidant, anti-carcinogenic, anti-tumor and immunomodulatory properties. However, it remains unclear whether cis-resveratrol may exhibit similar activities. The objective of the present study was to examine the effects of cis- and trans-resveratrol on the production of pro-inflammatory cytokines and mediators in human macrophages. We examined the possibility that cis- and trans-resveratrol may affect cytokine secretion by modulating inflammasomes, intracellular multi-protein complexes, the assembly of which leads to caspase-1 activation and secretion of active IL-1β by macrophages. Our results show that pre-treatment of macrophages with cis-resveratrol not only reduces pro-IL-1β production and IL-1β secretion, but also suppresses ATP-induced transcription and activation of caspase-1 and caspase-4. Notably, cis-resveratrol inhibits the expression of the purinergic receptor, P2X7R, and the endoplasmic reticulum stress marker, Glc-regulated protein 78, but also reduces reactive oxygen species production. Moreover, cis-resveratrol attenuates cyclooxygenase-2 expression and prostaglandin E2 production. cis-Resveratrol also decreases the phosphorylation of p38 MAPK and expression of the c-Jun protein. These results indicate that cis-resveratrol produces anti-inflammatory effects by inhibiting both the canonical and non-canonical inflammasomes, and associated pathways in human macrophages.

Hirsutella sinensis mycelium suppresses interleukin-1β and interleukin-18 secretion by inhibiting both canonical and non-canonical inflammasomes

Cordyceps sinensis is a medicinal mushroom used for centuries in Asian countries as a health supplement and tonic. Hirsutella sinensis—the anamorphic, mycelial form of C. sinensis—possesses similar properties, and is increasingly used as a health supplement. Recently, C. sinensis extracts were shown to inhibit the production of the pro-inflammatory cytokine IL-1β in lipopolysaccharide-treated macrophages. However, the molecular mechanism underlying this process has remained unclear. In addition, whether H. sinensis mycelium (HSM) extracts also inhibit the production of IL-1β has not been investigated. In the present study, the HSM extract suppresses IL-1β and IL-18 secretion, and ATP-induced activation of caspase-1. Notably, we observed that HSM not only reduced expression of the inflammasome component NLRP1 and the P2X7R but also reduced the activation of caspase-4, and ATP-induced ROS production. These findings reveal that the HSM extract has anti-inflammatory properties attributed to its ability to inhibit both canonical and non-canonical inflammasomes.

Inducible and constitutive HSP70s confer synergistic resistance against metabolic challenges

Chaperonic proteins, including inducible HSP70 (HSP70i) and constitutive HSP70 (HSC70), have been implicated as essential players in the cellular adaptive protection. Ensuing studies demonstrated that overexpression of either protein individually protects against thermal and oxidative challenges. The present study aimed to determine whether a concurrent overexpression of both HSC70 and HSP70i confers a better metabolic protection than the expression of each protein alone. Using a rat heart-derived H9c2 cardiac myoblast cell line, we found that HSP70i was rapidly induced within 2-8h following a mild thermal preconditioning (43°C for 20 min) in both parental cells and an established H9/70c clonal sub-line overexpressing HSC70. The level of HSP70i protein in heat pretreated H9/70c clonal cells reached only 50% of that in heat pretreated H9c2 parental cells. Nevertheless, protection against lethal hyperthermia, menadione (an oxidant) and hydrogen peroxide (H(2)O(2)) exposure in the pretreated H9/70c clonal cells was significantly higher than the sum of protection afforded by the early induction of HSP70i in the pretreated parental cells and protection afforded by the pre-existing HSC70 in the H9/70c cells without preconditioning. Using dosimetric analysis, we also found that menadione resistance in the pretreated parental cells increased linearly with cellular HSP70i level (10-300 ng/mg total protein). However, the resistance in the pretreated H9/70c cells showed a biphasic relationship with cellular HSP70i level; when HSP70i concentration reached >250 ng/mg protein, survivability after menadione exposure was markedly enhanced. Similar results were observed in H9c2 cells genetically manipulated to overexpress both HSC70 and HSP70i. The survival benefit against lethal hyperthermia, oxidant treatment, and hypoxia/reoxygenation conferred by a concerted HSC70 and HSP70i overexpression was greater than the sum of benefits contributed by individual protein overexpression. Together, these findings suggest that HSC70 and HSP70i may complement each other in a synergistic manner to preserve cellular integrity during metabolic challenges.

Transgenome transcription and replication in the liver and extrahepatic tissues of a human hepatitis B virus transgenic mouse

We have produced a transgenic mouse (B32-1) carrying the complete genome of the human hepatitis B virus (HBV). High titers of the viral surface (HBsAg) and the e antigen (HBeAg) were detected in the serum of the mouse. In the liver and 12 of 16 extrahepatic tissues analyzed, Northern blot hybridization indicated the presence of the 2.1-kilobase (kb) and the 3.5-kb major HBV transcripts. A liver cDNA library was constructed from which the liver RNAs from four cDNA clones with splicing were found. Sequencing analysis showed that the splicing occurred between nucleotides 2451 and 487 of the viral genome, resulting in a truncated viral polymerase gene, as in human hepatocytes. Southern blot analysis of total DNA preparations of the tissues revealed the presence of episomal HBV genome, indicating replication of the viral transgenome in these tissues. However, replication was detected only in some but not all of the tissues that transcribed the 3.5-kb RNA. Partial double-stranded as well as full-length and subgenomic-length single-stranded HBV DNA species of discrete sizes were detected which may represent replication intermediates of preferred replication termination sites of the HBV transgenome. Since many molecular characteristics of mouse B32-1 were similar to those found in HBV-infected humans, HBV transgenic mice similar to B32-1 would be useful in further elucidation of other aspects of the replication and transcription mechanisms of HBV in the liver and extrahepatic tissues.

FZD1 activates protein kinase C delta-mediated drug-resistance in multidrug-resistant MES-SA/Dx5 cancer cells

Multidrug-resistant (MDR) cancer is a major clinical problem in chemotherapy of cancer patients. We have noted inappropriate PKCδ hypomethylation and overexpression of genes in the PKCδ/AP-1 pathway in the human uterus sarcoma drug-resistant cell line, MES-SA/Dx5 cells, which also overexpress p-glycoprotein (ABCB1). Recent studies have indicated that FZD1 is overexpressed in both multidrug-resistant cancer cell lines and in clinical tumor samples. These data have led us to hypothesize that the FZD1-mediated PKCδ signal-transduction pathway may play an important role in drug resistance in MES-SA/Dx5 cells. In this work, the PKCδ inhibitor Rottlerin was found to reduce ABCB1 expression and to inhibit the MDR drug pumping ability in the MES-SA/Dx5 cells when compared with the doxorubicin-sensitive parental cell line, MES-SA. PKCδ was up-regulated with concurrent up-regulation of the mRNA levels of the AP-1-related factors, c-JUN and c-FOS. Activation of AP-1 also correlated with up-regulation of the AP-1 downstream genes HGF and EGR1. Furthermore, AP-1 activities were reduced and the AP-1 downstream genes were down-regulated in Rottlerin-treated or PKCδ shRNA-transfected cells. MES-SA/Dx5 cells were resensitized to doxorubicin-induced toxicity by co-treatment with doxorubicin and Rottlerin or PKCδ shRNA. In addition, cell viability and drug pump-out ability were significantly reduced in the FZD1 inhibitor curcumin-treated and FZD1 shRNA-knockdown MES-SA/Dx5 cells, indicating involvement of PKCδ in FZD1-modulated ABCB1 expression pathway. Taken together, our data demonstrate that FZD1 regulates PKCδ, and the PKCδ/AP-1 signalling transduction pathway plays an important role in drug resistance in MES-SA/Dx5 cells.

Disabled-2 is required for mesoderm differentiation of murine embryonic stem cells†

A variety of signaling networks are implicated in the control of mesoderm differentiation. Previous studies demonstrated that Disabled‐2 (DAB2) is a multifunctional protein involved in growth factor signaling and embryonic development. In this study, we investigated DAB2 expression and function during in vitro mesoderm differentiation of murine embryonic stem cells (ESCs). We found that DAB2 was up‐regulated when ESCs were co‐cultured with OP9 stromal cells for mesoderm differentiation. DAB2 was also up‐regulated when ESCs were induced for embryoid body formation. Expression of DAB2 short hairpin small interfering RNA (shDAB2) did not alter the puripotency of ESCs. However, shDAB2 disrupted ESCs cell‐cell adhesion and affected embryoid body and colony formation that subsequently impeded mesoderm differentiation of ESCs. Immunofluorescent staining revealed that disorganization of β‐catenin and plakoglobin cellular distribution may account for the aberrant cell–cell adhesion in DAB2‐deficient cells. Accordingly, DAB2 was identified as a plakoglobin‐binding partner with the interaction mediated by the phosphotyrosine binding domain of DAB2 and the Asn‐Pro‐Asp‐Tyr (NPDY) motif of plakoglobin. Molecular analysis and transcriptome profiling also revealed that DAB2 was involved in the regulation of insulin‐like growth factor 2‐mediated signaling and in the expression of p53, asparagine synthetase and glutathione peroxidase 2. Expression screening of 52 ESCs‐related miRNAs further unveiled the interplay between DAB2 and the signaling networks associated with cell death, differentiation and development. This study thereby defines a role of DAB2 in fate determination of ESCs and suggests the presence of a DAB2‐associated regulatory circuit in the control of mesoderm differentiation. J. Cell. Physiol. 225: 92–105, 2010.

Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.