Tryptophol Improves the Biocontrol Efficacy of Scheffersomyces spartinae against the Gray Mold of Strawberries by Quorum Sensing.

Previously, we reported that marine yeast Scheffersomyces spartinae exhibited biocontrol efficacy against the gray mold of strawberries caused by Botrytis cinerea. Herein, tryptophol, a quorum-sensing molecule, was identified in the metabolites of S. spartinae. Subsequently, we found that 25 µM tryptophol promoted population density, biofilm formation, and cell aggregation of S. spartinae. Furthermore, 25 µM tryptophol improved the biocontrol efficacy of S. spartinae against B. cinerea in vitro and in the strawberry fruit. Under a scanning electronic microscope, tryptophol facilitated colonization and biofilm formation on strawberry wounds, showing that tryptophol increased the biocontrol efficacy of S. spartinae via quorum sensing. Transcriptome analysis revealed that tryptophol upregulated the gene expression of SDS3, DAL81, DSE1, SNF5, SUN41, FLO8, and HOP1, which was associated with cell adhesion or biofilm formation. Thus, to the best of our knowledge, this study was the first to report that tryptophol improved the biocontrol efficacy of S. spartinae via quorum sensing.

ß-Glucan Enhances the Biocontrol Efficacy of Marine Yeast Scheffersomyeces spartinae W9 against Botrytis cinerea in Strawberries.

The marine yeast Scheffersomyeces spartinae W9 is a promising biocontrol agent for gray mold caused by Botrytis cinerea in strawberries. Improving the biocontrol efficacy of S. spartinae W9 is necessary for its commercial application. In this study, different concentrations of ß-glucan were added to the culture medium to evaluate its effect on the biocontrol efficacy of S. spartinae W9. The results showed that 0.1% ß-glucan could increase the biocontrol effect of S. spartinae W9 against B. cinerea in strawberries and in vitro. We found that adding 0.1% ß-glucan to the culture medium promoted the growth of S. spartinae W9 in wounds of strawberries, enhanced biofilm formation ability, and secreted more ß-1,3-glucanase. In addition, 0.1% ß-glucan increased the survival rate of S. spartinae W9 under oxidative, thermal, osmotic, and plasma membrane stressors. Transcriptome analysis revealed 188 differential expressed genes in S. spartinae W9 cultured with or without 0.1% ß-glucan, including 120 upregulated and 68 downregulated genes. The upregulated genes were associated with stress response, cell wall formation, energy production, growth, and reproduction. Thus, culturing with 0.1% ß-glucan is an effective way to improve the biocontrol ability of S. spartinae W9 against gray mold in strawberries.

Annexin A2 promotes development of retinal neovascularization through PI3K/ AKT signaling pathway.

PURPOSE: Retinal Neovascularization (RNV) is a pathological characteristic of ocular diseases. Annexin A2 (ANXA2) plays important roles in RNV while the mechanism remains unclear. The study aimed to explore relationship between ANXA2 and PI3K/AKT signaling pathway in RNV. METHODS: We used human retinal vascular endothelial cells (HRECs) and oxygen-induced retinopathy (OIR) mice model to show ANXA2 can promote the development of RNV through PI3K/AKT signaling pathway. We divided HRECs into six groups by infecting lentivirus containing appropriate plasmid and adding corresponding solution. Assays showing ability of HRECs were performed in vitro. Mice were randomly divided into three groups and treated accordingly. RESULTS: Expression of ANXA2 and activity of PI3K/AKT signaling pathway in HRECs were detected. RNV and expression of ANXA2 in mice retinas were detected. Results showed that ANXA2 expression is positively related with RNV-forming ability of HRECs in vitro and development of RNV in vivo while low activity of PI3K/AKT signaling pathway could attenuate the role of ANXA2. CONCLUSIONS: We can make ANXA2 and PI3K/ AKT signaling pathway as a promising target for the regulation of pathological neovascularization of the retina, which also provides a novel idea for effective prevention and treatment of diseases related to RNV in future.

Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes.

Exosomes, also known as extracellular vesicles, are naturally occurring, biocompatible, and bioacive nanoparticles ranging from 40 to 150 nm in diameter. Bone-secreted exosomes play important roles in bone homeostasis, the interruption of which can lead to diseases such as osteoporosis, rheumatoid arthritis, and osteopetrosis. Though the relationship between vascular and bone homeostasis has been recognized recently, the role of vascular endothelial cell (EC)-secreted exosomes (EC-Exos) in bone homeostasis is not well understood. Herein, we found that EC-Exos show more efficient bone targeting than osteoblast-derived exosomes or bone marrow mesenchymal stem cell-derived exosomes. We also found that EC-Exos can be internalized by bone marrow-derived macrophages (BMMs) to alter their morphology. EC-Exos can inhibit osteoclast activity in vitro and inhibit osteoporosis in an ovariectomized mouse model. Sequencing of exosome miRNA revealed that miR-155 was highly expressed in EC-Exos-treated BMMs. The miR-155 level in EC-Exos was much higher than that in BMMs and ECs, indicating that miR-155 was endogenous cargo of EC-derived vesicles. Blockage of BMMs miR-155 levels reversed the suppression by EC-Exos of osteoclast induction, confirming that exosomal miR-155 may have therapeutic potential against osteoporosis. Taken together, our findings suggest that EC-Exos may be utilized as a bone targeting and nontoxic nanomedicine for the treatment of bone resorption disorders.

Zoledronate inhibits fibroblasts' proliferation and activation via targeting TGF-ß signaling pathway.

BACKGROUND: Previous preclinical and clinical studies have demonstrated that zoledronate might inhibit neointimal hyperplasia at least partly by inhibiting the proliferation, adhesion and migration of vascular smooth muscle cells (VSMCs). However, whether zoledronate influences fibroblasts' proliferation and activation, which also play a key role in neointimal hyperplasia and vascular remodeling, remains largely unknown. In the present study, the effect of zoledronate on fibroblasts was investigated and the underlying molecular mechanisms were examined. METHODS: After treatment with zoledronate, changes in biological behaviors, including the morphology, proliferation, cell-cycle distribution and migration of fibroblasts (NIH3T3 cells), were observed. The expression of α-SMA, TGF-ß1 and TGF-ß2 and the level of Smad2/3 phosphorylation in cultured fibroblasts were examined by Western blot. In vivo expression of α-SMA and TGF-ß1 was assessed by immunohistochemical staining. RESULTS: It was shown that the typical fibroblast cell morphology was altered after zoledronate exposure. Cultured fibroblasts treated with zoledronate displayed dose-dependent inhibition of cell proliferation due to cell-cycle arrest in the S phase. Cell migration activities were also dose dependently suppressed by zoledronate treatment. Expression of α-SMA in cultured fibroblasts was significantly reduced by zoledronate treatment. Further analysis showed decreased expression of TGF-ß1 and α-SMA by periadventitial delivery of zoledronate in the rat carotid balloon-injury model. The expression of TGF-ß1 and TGF-ß2 and the phosphorylation of Smad2/3 in cultured fibroblasts were significantly inhibited by zoledronate treatment. CONCLUSION: Our findings demonstrated that zoledronate can inhibit the proliferation, migration and activation of fibroblasts via the TGF-ß signaling pathway and revealed a novel mechanism of zoledronate action against neointimal hyperplasia.

Biocompatible PEGylated Gold nanorods function As cytokinesis inhibitors to suppress angiogenesis.

Pathological angiogenesis is driven by uncontrolled growth of endothelial cells (ECs), which could lead to retinopathy, tumor and rheumatoid arthritis, etc. ECs must experience multiple cell division process to grow, and cytokinesis is the final step. The present study shows that PEGylated GNRs (PEG-GNRs) specifically target ECs cytokinesis process which results in high ratio of binucleated cells, and these binucleated ECs lose the ability to proliferate. Further data show that PEG-GNRs do not induce toxicity in vitro and in vivo. PEG-GNRs could inhibit ECs proliferation, migration, tube formation and inhibit angiogenesis in ex vivo model. Oxygen induced retinopathy and tumor angiogenesis model further show that PEG-GNRs can inhibit angiogenesis in vivo. Gene expression profiles reveal that PEG-GNRs mainly affect ECs cell division process, and PEG-GNRs treated ECs are arrested in G2/M phase. The mechanism is that PEG-GNRs could disrupt TGFß pathway, and subsequently suppress the assembly of actin filaments in contractile ring site. These findings indicate that PEG-GNR is a novel cytokinesis inhibitor which can be used to interfere with retinal angiogenesis and tumor.

Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2.

BACKGROUND/AIMS: Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. METHODS: We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. RESULTS: Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2. CONCLUSIONS: Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future.