Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells.

Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-ß1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

Cytochrome P450 Mediated Bioactivation of Rutaevin, a Bioactive and Potentially Hepatotoxic Component of Evodia Rutaecarpa.

Rutaevin is one of the major bioactive constituents isolated from Evodia rutaecarpa, a well-known herbal medicine that has been widely prescribed for the treatment of gastrointestinal disorders in China. However, oral administration of rutaevin has been shown to cause hepatotoxicity in mice. Bioactivation was suggested to be involved in rutaevin-induced hepatotoxicity. The aim of this study was to investigate the bioactivation of rutaevin in rat and human liver microsomes fortified with NADPH. Rutaevin was metabolized into the reactive intermediate cis-butene-1,4-dial (BDA) that was dependent on NADPH. The rutaevin-derived BDA intermediate was trapped by nucleophiles such as glutathione (GSH), N-acetyl-lysine (NAL), and methoxylamine (MOA) in the microsomal incubation system. A total of 10 conjugates resulting from the conjugation of the intermediate with GSH, NAL, or MOA were detected and structurally characterized by liquid chromatography combined with high-resolution tandem mass spectrometry. M1, structurally confirmed by NMR spectroscopic analysis, was identified as a cyclic mono(GSH) conjugate of the BDA intermediate, which was also found in the biliary samples of rutaevin-treated rats. Further inhibitory experiments suggested that ketoconazole showed strong inhibitory effect on the formation of the rutaevin-derived BDA intermediate. CYP3A4 was demonstrated to be the major enzyme responsible for rutaevin bioactivation by using cDNA-expressed human recombinant cytochrome P450 enzymes. Additionally, it was found that rutaevin was a mechanism-based inactivator of CYP3A4, with inactivation parameters of KI = 15.98 µM, kinact = 0.032 min-1, and t1/2 inact = 21.65 min. In summary, these findings are of great significance in understanding the bioactivation mechanism of rutaevin, the potential mechanism of rutaevin-caused hepatotoxicity, and the drug-drug interactions associated with rutaevin mainly via CYP3A4 inactivation.

Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7.

The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections.IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

Tetramethylpyrazine protects against high glucose-induced vascular smooth muscle cell injury through inhibiting the phosphorylation of JNK, p38MAPK, and ERK.

Objectives High glucose-induced alterations in vascular smooth muscle cell behavior have not been fully characterized. We explored the protective mechanism of tetramethylpyrazine (TMP) on rat smooth muscle cell injury induced by high glucose via the mitogen-activated protein kinase (MAPK) signaling pathway. Methods Vascular smooth muscle cells (VSMCs) isolated from rat thoracic aortas were divided into control, high glucose (HG), and pre-hatching TMP groups. The effect of different glucose concentrations on cell viability and on the migration activity of VSMC cells was examined using MTT analysis and the wound scratch assay, respectively. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using enzyme-linked immunoassays. The levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38MAPK, and MAPK phosphorylation were assessed by western blotting. Results Cell proliferation was remarkably increased by increased glucose concentrations. Compared with the HG group, the migratory ability of VSMC cells was reduced in the presence of TMP. TMP also decreased the MDA content in the supernatant, but significantly increased the SOD activity. Western blotting showed that TMP inhibited the phosphorylation of JNK, p38MAPK, and ERK. Conclusions TMP appears to protect against HG-induced VSMC injury through inhibiting reactive oxygen species overproduction, and p38MAPK/JNK/ERK phosphorylation.

Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro.

Cardiac mesenchymal stem cells (C-MSCs) are endogenous cardiac stromal cells that play a role in heart repair after injury. C-MSC-derived exosomes (Exo) have shown protective effects against apoptosis induced by acute myocardial ischemia/reperfusion. Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine (TCM) formula used in China for the treatment of acute myocardial ischemia, which contains tetramethylpyrazine (TMP) and borneol (BOR) as major components. In this study, we investigated whether SJP treatment affected exosome release from C-MSCs in vitro. C-MSCs prepared from mice were treated with SJP (62.5 µg/mL), TMP (25 µg/mL) or BOR (15 µg/mL). Using an acetylcholinesterase activity assay, we found that both SJP and TMP treatment significantly increased exosome secretion compared to the control ethanol treatment. The neutral sphingomyelinase 2 (nSMase2) pathway was important in exosome formation and packaging. But neither the level of nSMase2 mRNA nor the level of protein changed following SJP, TMP or BOR treatment, suggesting that SJP stimulated exosome release via an nSMase2-independent pathway. The Rab27a and Rab27b GTPases controlled different steps of the exosome secretion pathway. We showed that SJP treatment significantly increased the protein levels of Rab27a, SYTL4 (Rab27a effector) and Rab27b compared with the control treatment. SJP treatment also significantly upregulated the mRNA level of Rab27b, rather than Rab27a. Moreover, SJP-induced increase of C-MSC-exosome release was inhibited by Rab27b knockdown, suggesting that SJP promotes exosome secretion from C-MSCs via a GTPase-dependent pathway. This study reveals a novel mechanism for SJP in modulating cardiac homeostasis.

Exosomes from Suxiao Jiuxin pill-treated cardiac mesenchymal stem cells decrease H3K27 demethylase UTX expression in mouse cardiomyocytes in vitro.

Suxiao Jiuxin pill (SJP) is a traditional Chinese medicine for the treatment of acute coronary syndrome in China, which contains two principal components, tetramethylpyrazine (TMP) and borneol (BOR). Thus far, however, the molecular mechanisms underlying the beneficial effects of SJP on the cardiac microenvironment are unknown. Cardiac mesenchymal stem cells (C-MSCs) communicate with cardiomyocytes (CMs) through the release of microvesicles (exosomes) to restore cardiac homeostasis and elicit repair, in part through epigenetic regulatory mechanisms. In this study, we examined whether SJP treatment altered C-MSC-derived exosomes (SJP-Exos) to cause epigenetic chromatic remodeling in recipient CMs. C-MSC isolated from mouse hearts were pretreated with SJP (SJP-Exos), TMP (TMP-Exos) or BOR (BOR-Exos). Then, HL-1 cells, a mouse cardiomyocyte line, were treated with exosomes from control C-MSCs (Ctrl-Exos), SJP-Exos, TMP-Exos or BOR-Exos. Treatment with SJP-Exos significantly increased the protein levels of histone 3 lysine 27 trimethylation (H3K27me3), a key epigenetic chromatin marker for cardiac transcriptional suppression, in the HL-1 cells. To further explore the mechanisms of SJP-Exo-mediated H3K27me3 upregulation, we assessed the mRNA expression levels of key histone methylases (EZH1, EZH2 and EED) and demethylases (JMJD3 and UTX) in the exosome-treated HL-1 cells. Treatment with SJP-Exo selectively suppressed UTX expression in the recipient HL-1 cells. Furthermore, PCNA, an endogenous marker of cell replication, was significantly higher in SJP-Exo-treated HL-1 cells than in Ctrl-Exo-treated HL-1 cells. These results show that SJP-Exos increase cardiomyocyte proliferation and demonstrate that SJP can modulate C-MSC-derived exosomes to cause epigenetic chromatin remodeling in recipient cardiomyocytes; consequently, SJP-Exos might be used to promote cardiomyocyte proliferation.

Adjuvant chemotherapy after esophagectomy: is there a role in the treatment of the lymph node positive thoracic esophageal squamous cell carcinoma?

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) patients with regional lymph node metastases have poor prognosis after surgery. The purpose of this study was to investigate the impact of various treatment modalities on survival in these patients. METHODS: We retrospectively reviewed data from 349 patients who had undergone left transthoracic esophagectomy for thoracic ESCC from January 2008 to December 2010 at our institute. All patients had lesions in the mid or lower third of the thoracic segment and had pathological positive lymph node metastasis. Of these patients, 143 patients received surgery alone, 154 patients underwent postoperative radiotherapy alone, and 52 patients underwent taxane-based chemotherapy. Univariate and multivariate Cox regression analyses were used to analyze prognostic factors and survival. RESULTS: At a median follow-up of 53.1 months, the 3-year OS were 47.7% for the patients with surgery alone, 44.0% for the patients with adjuvant radiotherapy, and 58.9% for the patients with adjuvant chemotherapy. Multivariate analysis showed that postoperative therapy with adjuvant chemotherapy was significant positive predictor of survival. CONCLUSIONS: Postoperative taxane-based adjuvant chemotherapy improved survival of patients with lymph node positive thoracic ESCC compared with surgery alone. Further randomized prospective studies to confirm these findings are warranted.

[Randomize trial of cisplatin plus gemcitabine with either sorafenib or placebo as first-line therapy for non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: Platinum-based chemotherapy doublets reached an efficacy plateau in nonsmall-cell lung cancer (NSCLC). This randomized controlled study prospectively assessed the efficacy and safety of cisplatin plus gemcitabine with either Sorafenib or placebo as first-line therapy for NSCLC. METHODS: Thirty patients, which were confirmed advanced NSCLC histologically or cytologically, were randomly assigned to receive up to six cycles of cisplatin plus gemcitabine with sorafenib or placebo. The maintenance of sorafenib or placebo after chemotherapy will continued in patients with response or stable disease until disease progression or unacceptable adverse events. RESULTS: Overall demographics were balanced between experimental group (sorafenib+chemotherapy) and controlled group (chemotherapy only). Overall response (OS) rate was 55.6% and 41.7% in experimental arm and controlled arm, respectively (P=0.905). Median progressive-free survival (PFS) and median overall survival were similar (5 months vs 4 months, P=0.75; 18 months vs 18 months, P=0.68). Adverse events were tolerable, though the risk of hypertension and diarrhea was increase in experimental arm. Since patients with ECOG PS 0, stage IIIb, no liver metastasis and tyrasine kinasis inhibitor treatment after study had longer survive, these factors seemed to be predictive factors favor of survival in Cox regression analyses. CONCLUSIONS: No additional benefit of response rate, PFS or OS were observed from adding targeted agent-sorafenib to regular cisplatin plus gemcitabine chemotherapy. Selecting aproper patients is needed in further study.

Formulation of Docetaxel by folic acid-conjugated d-α-tocopheryl polyethylene glycol succinate 2000 (Vitamin E TPGS(2k)) micelles for targeted and synergistic chemotherapy.

Although high efficacy has been showed, Paclitaxel and Docetaxel cause serious side effects due to the adjuvant used in their clinical formulation Taxol® and Taxotere®. We developed a micelle system with a newly synthesized TPGS(2k) polymer, which shows lower CMC of 0.0219 mg/ml compared with 0.2 mg/ml for traditional micelles with TPGS involved, to achieve sustained and controlled drug delivery with Docetaxel used as a model anti-cancer drug. The TPGS(2k) micelles were further conjugated to folic acid (FA) for targeted drug delivery. The Docetaxel-loaded TPGS(2k) micelles with and without FA conjugation were found of desired size and size distribution, high drug encapsulation efficiency and favorable drug release. In vitro studies using MCF-7 cancer cells demonstrated significantly the higher cellular uptake of the formulated drug for TPGS(2k) micelle formulation than that for Taxotere®. The targeting effects for the FA conjugated TPGS(2k) micelles are also demonstrated. The IC50 value, which is the drug concentration needed for 50% cell viability in the designated time period, is 103.4, 1.280 and 0.1480 µg/ml for MCF-7 cancer cells after 24, 48, and 72 h treatment respectively, which is greatly decreased to be 0.526, 0.251 and 0.233 µg/ml, i.e. a 99.5%, 80.4% decrease and 57.5% increase for the TPGS(2k) micelle formulation, and further decreased to be 0.1780, 0.1520 and 0.1140 µg/ml, i.e. a 99.8%, 88.1% and 23.0% decrease for the folic acid conjugated micelles, respectively. A synergistic effect between TPGS(2k) and Docetaxel is also achieved. The present work represents a new concept in the design of drug delivery systems--the carrier materials of the drug delivery system can also have therapeutic effects, which either modulate the side effects of, or promote a synergistic interaction with the formulated drug.