Low-GDP, pH-neutral solutions preserve peritoneal endothelial glycocalyx during long-term peritoneal dialysis.

BACKGROUND: During peritoneal dialysis (PD), solute transport and ultrafiltration are mainly achieved by the peritoneal blood vasculature. Glycocalyx lies on the surface of endothelial cells and plays a role in vascular permeability. Low-glucose degradation product (GDP), pH-neutral PD solutions reportedly offer higher biocompatibility and lead to less peritoneal injury. However, the effects on the vasculature have not been clarified. METHODS: Peritoneal tissues from 11 patients treated with conventional acidic solutions (acidic group) and 11 patients treated with low-GDP, pH-neutral solutions (neutral group) were examined. Control tissues were acquired from 5 healthy donors of kidney transplants (control group). CD31 and ratio of luminal diameter to vessel diameter (L/V ratio) were evaluated to identify endothelial cells and vasculopathy, respectively. Immunostaining for heparan sulfate (HS) domains and Ulex europaeus agglutinin-1 (UEA-1) binding was performed to assess sulfated glycosaminoglycans and the fucose-containing sugar chain of glycocalyx. RESULTS: Compared with the acidic group, the neutral group showed higher CD31 positivity. L/V ratio was significantly higher in the neutral group, suggesting less progression of vasculopathy. Both HS expression and UEA-1 binding were higher in the neutral group, whereas HS expression was markedly more preserved than UEA-1 binding in the acidic group. In vessels with low L/V ratio, which were found only in the acidic group, HS expression and UEA-1 binding were diminished, suggesting a loss of glycocalyx. CONCLUSION: Peritoneal endothelial glycocalyx was more preserved in patients treated with low-GDP, pH-neutral solution. The use of low-GDP, pH-neutral solutions could help to protect peritoneal vascular structures and functions.

Long Noncoding RNA HOTAIR Promotes Epithelial-Mesenchymal Transition and Is a Suitable Target to Inhibit Peritoneal Dissemination in Human Scirrhous Gastric Cancers.

OBJECTIVES: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer. METHODS: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination. RESULTS: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination - and significantly prolonged the survival - in the orthotopic tumor mouse model. CONCLUSION: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.

Excessive salt intake increases peritoneal solute transport rate via local tonicity-responsive enhancer binding protein in subtotal nephrectomized mice.

BACKGROUND: High peritoneal transport is associated with high mortality and technical failure in peritoneal dialysis (PD). Baseline peritoneal solute transport rate (PSTR) as measured by the peritoneal equilibration test (PET) within 6 months after PD initiation varies between patients. Sodium is reported to be stored in the skin or muscle of dialysis patients. This study investigated whether excessive salt intake in uremic mice caused peritoneal alterations without exposure to PD fluid. METHODS: Sham-operated (Sham) and subtotal nephrectomized (Nx) mice were randomly given tap water or 1% sodium chloride (NaCl)-containing water for 8 weeks. PET was then performed to evaluate peritoneal function. Human mesothelial cell line Met-5A was used for in vitro studies. RESULTS: We observed higher PSTR in Nx mice with 1% NaCl-containing drinking water (Nx + salt) compared with those with tap water (Nx + water), along with enhanced angiogenesis and inflammation in the peritoneum. Blockade of interleukin (IL)-6 signaling rescued peritoneal transport function in Nx + salt mice. In cultured Met-5A, additional NaCl in the medium upregulated IL-6 as well as vascular endothelial growth factor-A, associated with increased expression and nuclear translocation of tonicity-responsive enhancer binding protein (TonEBP). Knockdown of TonEBP lowered the induction caused by high tonicity. Peritoneal TonEBP expression was higher in Nx + salt mice, while removal of high-salt diet lowered TonEBP level and improved peritoneal transport function. CONCLUSIONS: Excessive dietary salt intake caused peritoneal membrane functional and structural changes under uremic status. TonEBP regulated hypertonicity-related inflammatory changes and might play a crucial role in high baseline peritoneal transport.

Effects of long-term treatment with low-GDP, pH-neutral solutions on peritoneal membranes in peritoneal dialysis patients.

BACKGROUND: The morphological changes induced by bio-incompatible peritoneal dialysis (PD) solutions are well known. However, the morphological damage induced by long-term low-glucose degradation product (GDP), pH-neutral solutions has not been reported in detail. The aim of this study was to investigate the long-term effects of pH-neutral PD solutions on morphological and functional changes in the peritoneal membrane. METHODS: We assessed peritoneal membrane biopsy samples from PD patients treated with acidic (Conventional group) or pH-neutral solutions (pH-neutral group) using pathology and immunopathology techniques. RESULTS: Analyses of 54 Conventional and 73 pH-neutral group samples showed that the peritoneal membrane was thicker (P < 0.001), the ratio of luminal diameter to vessel diameter (L/V ratio) was significantly smaller (P < 0.001), and advanced glycation end-product (AGE) accumulation was higher in the Conventional than in the pH-neutral group (P < 0.001). Comparison of samples from patients in the Conventional (n = 33) and pH-neutral groups (n = 33) who were treated for 4-10 years also showed significant differences in peritoneal thickness, L/V ratio and AGE score. Furthermore, the L/V ratio in the Conventional group significantly decreased over time (P < 0.01); however, no such change was seen in the pH-neutral group. Peritoneal membrane thickness was not associated with PD duration in both groups. Dialysate-to-plasma ratio of creatinine and L/V ratio negatively correlated with PD treatment duration in the Conventional group, but not in the pH-neutral group. CONCLUSIONS: These findings suggest that pH-neutral solutions prevent the morphological and functional peritoneal changes induced by long-term PD treatment.

TGF-ß1-VEGF-A pathway induces neoangiogenesis with peritoneal fibrosis in patients undergoing peritoneal dialysis.

The characteristic features of chronic peritoneal injury with peritoneal dialysis (PD) are submesothelial fibrosis and neoangiogenesis. Transforming growth factor (TGF)ß and vascular endothelial growth factor (VEGF)-A are the main mediators of fibrosis and neoangiogenesis, respectively; however, the effect of the interaction between them on the peritoneum is not well known. In this study, we investigated the relationship between TGF-ß1 and VEGF-A in inducing peritoneal fibrosis by use of human tissues and dialysate, cultured cells, and animal models. The VEGF-A concentration correlated with the dialysate-to-plasma ratio of creatinine (D/P Cr) ( P < 0.001) and TGF-ß1 ( P < 0.001) in human PD effluent. VEGF-A mRNA levels increased significantly in the peritoneal tissues of human ultrafiltration failure (UFF) patients and correlated with number of vessels ( P < 0.01) and peritoneal thickness ( P < 0.001). TGF-ß1 increased VEGF-A production in human mesothelial cell lines and fibroblast cell lines, and TGF-ß1-induced VEGF-A was suppressed by TGF-ß receptor I (TGFßR-I) inhibitor. Incremental peak values of VEGF-A mRNA stimulated by TGF-ß1 in human cultured mesothelial cells derived from PD patients with a range of peritoneal membrane functions correlated with D/P Cr ( P < 0.05). To evaluate the regulatory mechanisms of VEGF-A and neoangiogenesis in vivo, we administered TGFßR-I inhibitor intraperitoneally in a rat chlorhexidine-induced peritoneal injury (CG) model. TGFßR-I inhibitor administration in the CG model decreased peritoneal thickness ( P < 0.001), the number of vessels ( P < 0.001), and VEGF-A levels ( P < 0.05). These results suggest that neoangiogenesis is associated with fibrosis through the TGF-ß1-VEGF-A pathway in mesothelial cells and fibroblasts. These findings are important when considering the strategy for management of UFF in PD patients.

MicroRNAs Associated with Epithelial-Mesenchymal Transition Can Be Targeted to Inhibit Peritoneal Dissemination of Human Scirrhous Gastric Cancers.

OBJECTIVES: Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. METHODS: We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. RESULTS: miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. CONCLUSIONS: EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers.

Roles of the TGF-ß⁻VEGF-C Pathway in Fibrosis-Related Lymphangiogenesis.

Lymphatic vessels drain excess tissue fluids to maintain the interstitial environment. Lymphatic capillaries develop during the progression of tissue fibrosis in various clinical and pathological situations, such as chronic kidney disease, peritoneal injury during peritoneal dialysis, tissue inflammation, and tumor progression. The role of fibrosis-related lymphangiogenesis appears to vary based on organ specificity and etiology. Signaling via vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor (VEGFR)-3 is a central molecular mechanism for lymphangiogenesis. Transforming growth factor-ß (TGF-ß) is a key player in tissue fibrosis. TGF-ß induces peritoneal fibrosis in association with peritoneal dialysis, and also induces peritoneal neoangiogenesis through interaction with VEGF-A. On the other hand, TGF-ß has a direct inhibitory effect on lymphatic endothelial cell growth. We proposed a possible mechanism of the TGF-ß⁻VEGF-C pathway in which TGF-ß promotes VEGF-C production in tubular epithelial cells, macrophages, and mesothelial cells, leading to lymphangiogenesis in renal and peritoneal fibrosis. Connective tissue growth factor (CTGF) is also involved in fibrosis-associated renal lymphangiogenesis through interaction with VEGF-C, in part by mediating TGF-ß signaling. Further clarification of the mechanism might lead to the development of new therapeutic strategies to treat fibrotic diseases.

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3.

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 µM siRNA, 25 µl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Sodium chloride promotes tissue inflammation via osmotic stimuli in subtotal-nephrectomized mice.

Chronic inflammation, which is often associated with high all-cause and cardiovascular mortality, is prevalent in patients with renal failure; however, the precise mechanisms remain unclear. High-salt intake was reported to induce lymphangiogenesis and autoimmune diseases via osmotic stimuli with accumulation of sodium or chloride. In addition, sodium was recently reported to be stored in the extremities of dialysis patients. We studied the effects and mechanisms of high salt loading on tissue and systemic inflammation in subtotal-nephrectomized mice (5/6Nx) and in cultured cells. Macrophage infiltration in the peritoneal wall (P<0.001), heart (P<0.05) and para-aortic tissues (P<0.001) was significantly higher in 5/6Nx with salt loading (5/6Nx/NaCl) than in 5/6Nx without salt loading (5/6Nx/Water); however, there were no significant differences in blood pressure and renal function between the groups. Tissue interleukin-6, monocyte chemotactic protein-1 (MCP-1), serum- and glucocorticoid-inducible kinase 1 (Sgk1) and tonicity-responsive enhancer binding protein (TonEBP) mRNA were significantly elevated in the peritoneal wall and heart with 5/6Nx/NaCl when compared with 5/6Nx/Water. Sodium was stored in the abdominal wall, exerting high-osmotic conditions. Reversal of salt loading reduced macrophage infiltration associated with decreased TonEBP in 5/6Nx/NaCl. Macrophage infiltration associated with fibrosis induced by salt loading was decreased in the 5/6Nx/NaCl/CC chemokine receptor 2 (CCR2, receptor of MCP-1)-deficient mice when compared with 5/6Nx/NaCl/Wild mice, suggesting that CCR2 is required for macrophage infiltration in 5/6Nx with NaCl loading. In cultured mesothelial cells and cardiomyocytes, culture media with high NaCl concentration induced MCP-1, Sgk1 and TonEBP mRNA, all of which were suppressed by TonEBP siRNA, indicating that both MCP-1 and Sgk1 are downstream of TonEBP. Our study indicates that high NaCl intake induces MCP-1 expression leading to macrophage infiltration via the TonEBP-MCP-1 pathway in 5/6Nx/NaCl mice, and that TonEBP has a central role in inflammation in patients with renal failure taking high salt.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-ß1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

TGF-ß1 promotes lymphangiogenesis during peritoneal fibrosis.

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-ß1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-ß1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-ß type I receptor (TGFßR-I) inhibitor. TGF-ß1-induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFßR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-ß-VEGF-C pathway.

Atelocollagen-mediated systemic delivery prevents immunostimulatory adverse effects of siRNA in mammals.

Short interfering RNA (siRNA) is a potent activator of the mammalian innate immune system. When considering possible clinical applications of siRNA for humans, the adverse immunostimulatory effects must also be taken into account. Here, we show that atelocollagen-mediated systemic delivery of siRNA without chemical modifications did not cause any immunostimulation in both animals and human peripheral blood mononuclear cells (PBMCs), even if the siRNA harbored an interferon (IFN)-inducible sequence. In contrast, systemic delivery of immunostimulatory RNA (isRNA)-mediated by a cationic lipid (such as Invivofectamine) induced potent type-I IFNs and inflammatory cytokines. Regarding the mechanism by which the isRNA/atelocollagen complex avoided adverse effects on immunostimulation, we revealed that this complex was not incorporated into PBMCs. On the other hand, Invivofectamine delivered isRNA into PBMCs. The use of either atelocollagen or Invivofectamine as a vehicle elicited significant and undistinguishable therapeutic effects in a contact hypersensitivity (CHS) inflammatory model mouse, when we intravenously injected the siRNA targeting monocyte chemoattractant protein-1 as the complex. For the goal of realizing siRNA-based medicines for humans, atelocollagen is an excellent and promising delivery vehicle, and it has the useful advantage of evading detection by the "radar" of innate immunity.

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1.

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.

Overexpression of BMI-1 correlates with drug resistance in B-cell lymphoma cells through the stabilization of survivin expression.

The expression of BMI-1 is correlated with disease progression in cancer patients. We showed that ectopic expression of BMI-1 in B-cell lymphoma cell lines, HT and RL, conferred resistance to etoposide and oxaliplatin, known to enhance sensitivity by targeting the survivin gene, but not to irinotecan, which is not relevant to the downregulation of survivin expression. The expression of survivin was not only augmented in cells transduced with BMI-1, but persisted in the presence of etoposide in cells overexpressing BMI-1. By contrast, the mock-transduced cells succumbed in the medium with anticancer drugs, with an accompanying decrease in BMI-1 and survivin expression. BMI-1 overexpression stabilized survivin post-translationally without an accompanying rise in the mRNA, suggesting survivin as a potential target for BMI-1. Knockdown of either BMI-1 or survivin restored sensitivity to etoposide in the BMI-1-overexpressing lymphoma cells. An analysis of six patients with B-cell lymphoma showed that in the drug-resistant patients, levels of BMI-1 and survivin were maintained even after drug administration. However, downregulation of both BMI-1 and survivin expression was observed in the drug-sensitive patients. Therefore, BMI-1 might facilitate drug resistance in B-cell lymphoma cells through the regulation of survivin. BMI-1 could be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas.

Transforming growth factor-ß induces vascular endothelial growth factor-C expression leading to lymphangiogenesis in rat unilateral ureteral obstruction.

Inflammation is recognized as an important contributor to lymphangiogenesis; however, in tubulointerstitial lesions in human chronic kidney diseases, this process is better correlated with the presence of myofibroblasts rather than macrophages. As little is known about the interaction between lymphangiogenesis and renal fibrosis, we utilized the rat unilateral ureteral obstruction model to analyze inflammation, fibrosis, lymphangiogenesis, and growth factor expression. Additionally, we determined the relationship between vascular endothelial growth factor-C (VEGF-C), an inducer of lymphangiogenesis, and the profibrotic factor, transforming growth factor-ß1 (TGF-ß1). The expression of both TGF-ß1 and VEGF-C was detected in tubular epithelial and mononuclear cells, and gradually increased, peaking 14 days after ureteral obstruction. The kinetics and localization of VEGF-C were similar to those of TGF-ß1, and the expression of these growth factors and lymphangiogenesis were linked with the progression of fibrosis. VEGF-C expression was upregulated by TGF-ß1 in cultured proximal tubular epithelial cells, collecting duct cells, and macrophages. Both in vitro and in vivo, the induction of VEGF-C along with the overall appearance of lymphatics in vivo was specifically suppressed by the TGF-ß type I receptor inhibitor LY364947. Thus, TGF-ß1 induces VEGF-C expression, which leads to lymphangiogenesis.

SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity.

Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

Amino-modified and lipid-conjugated dicer-substrate siRNA enhances RNAi efficacy.

The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of amino-modified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5'- and/or 3'-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3'-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3'-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.

Lipid-conjugated 27-nucleotide double-stranded RNAs with dicer-substrate potency enhance RNAi-mediated gene silencing.

Short interfering RNAs (siRNAs), used in RNA interference (RNAi) technology, are powerful tools for target-gene silencing in a sequence-specific manner. In this study, Dicer-substrate 27-nucleotide (nt) double-stranded RNAs (dsRNAs), which are known to have a highly potent RNAi effect, were conjugated with palmitic acid at the 5'-end of the sense strand to enhance intracellular delivery and RNAi efficacy. The palmitic acid-conjugated 27-nt dsRNAs (C16-ds27RNAs) were prepared by our simple synthesis strategy in good yield. The C16-ds27RNAs were cleaved by a Dicer enzyme, leading to the release of 21-nt siRNAs. The high level of stability in serum using C16-ds27RNAs was also confirmed. The C16-ds27RNAs showed enhanced RNAi potency targeted to both an exogenous luciferase and an endogenous vascular endothelial growth factor (VEGF) gene in the presence or absence of a transfection reagent, such as Lipofectamine 2000. In addition, the C16-ds27RNAs had a more potent gene-silencing activity than the other lipid-conjugated 21-nt siRNAs and 27-nt dsRNAs. The C16-ds27RNAs also exhibited significant membrane permeability. These results suggested that the C16-ds27RNAs will be useful for next-generation RNAi molecules that can address the problems of RNAi technology.

Establishment and optimal culture conditions of microrna-induced pluripotent stem cells generated from HEK293 cells via transfection of microrna-302s expression vector.

Induced pluripotent stem cells (iPSCs) have been directly generated from fibroblast cultures though retrovirus- or lentivirus-mediated ectopic overexpression of only a few defined transcriptional factors. This remarkable achievement has greatly enhanced our ability to explore the causes of, and potential cures for, many genetic diseases, and strengthened the promise of regenerative medicine. In fact, to date, many kinds of somatic cells from different tissues have exhibited a capacity for reprogramming toward an embryonic stem cell-like state, but major bottlenecks in iPSC derivation and therapeutic use remain, including low reprogramming efficiencies and the tumorigenesis of the generated iPSC. Here, we successfully generated miR-302s-induced pluripotent stem cells (mirPS cells) from human embryonic kidney (HEK) 293 cells via transfection of the miR-302s expression vector. We also determined the optimal culture conditions to generate mirPS on feeder cells, which included the use of serum-free N2B27 medium. The mirPS cells generated by our improved conditions showed the expression of pluripotent marker genes such as OCT3/4, NANOG, and SOX2 under growth conditions via reverse transcription-PCR, whereas no expression of these genes was observed in HEK293 cells. On the other hand, under differentiation conditions, mirPS cells formed ball-shaped structures (embryoid bodies), and showed the ability to differentiate into three germ layers (ectoderm, mesoderm, and endoderm) in vitro. The results suggested that our generated mirPS cells are actually functional as a cell resource to apply to regenerative medicine, and mirPS cells are suitable materials to clarify the mechanism underlying the reprogramming from somatic cells.