TNF-α-induced exosomal miR-146a mediates mesenchymal stem cell-dependent suppression of urethral stricture

The effective treatment of urethral stricture remains a medical problem. The use of proinflammatory cytokines as stimuli to improve the reparative efficacy of mesenchymal stem cells (MSCs) towards damaged tissues represents an evolving field of investigation. However, the therapeutic benefits of this strategy in the treatment of urethral stricture remain unknown. Here, we enriched exosomes derived from human umbilical cord-derived MSCs pretreated with or without tumor necrosis factor alpha (TNF-α) to evaluate their therapeutic effects in an in vivo model of TGFβ1-induced urethral stricture. Male Sprague-Dawley rats received sham (saline) or TGFβ1 injections to urethral tissues followed by incisions in the urethra. Animals in the TGFβ1 injection (urethral fibrosis) cohort were subsequently injected with vehicle control, or with exosomes derived from MSCs cultured with or without TNF-α. After 4 weeks, rats underwent ultrasound evaluation and, following euthanasia, urethral tissues were harvested for histological and molecular analysis. In vitro, the effects of MSC-derived exosomes on fibroblast secretion of collagen and cytokines were studied by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Exosomes derived from MSCs pretreated with TNF-α were more effective in suppressing urethral fibrosis and stricture than exosomes from untreated MSCs. We found that miR-146a, an anti-inflammatory miRNA, was strongly upregulated in TNF-α-stimulated MSCs and was selectively packaged into exosomes. Moreover, miR-146a-containing exosomes were taken up by fibroblasts and inhibited fibroblast activation and associated inflammatory responses, a finding that may underlie the therapeutic mechanism for suppression of urethral stricture. Inhibition of miR-146a in TNF-α-treated MSCs partially reduced antifibrotic effects and increased the release of proinflammatory factors of exosomes derived from these cells. Together these findings demonstrate that exosomes derived from TNF-α-treated MSCs are of therapeutic benefit in urethral fibrosis, suggesting that this strategy may have utility as an adjuvant therapy in the treatment of urethral stricture diseases.     

Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator–activated receptor-γ coactivator-1α

In varicose veins, vascular smooth muscle cells (VSMCs) often show abnormal proliferative and migratory rates and phenotypic transition. This study aimed to investigate whether microRNA (miR)-202 and its potential target, peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), were involved in VSMC phenotypic transition. miR-202 expression was analyzed in varicose veins and in VSMCs conditioned with platelet-derived growth factor. The effect of miR-202 on cell proliferation and migration was assessed. Furthermore, contractile marker SM-22α, synthetic markers vimentin and collagen I, and PGC-1α were analyzed by Western blot analysis. The modulation of PGC-1α expression by miR-202 was also evaluated. In varicose veins and proliferative VSMCs, miR-202 expression was upregulated, with decreased SM-22α expression and increased vimentin and collagen I expression. Transfection with a miR-202 mimic induced VSMC proliferation and migration, whereas a miR-202 inhibitor reduced cell proliferation and migration. miR-202 mimic constrained luciferase activity in HEK293 cells that were cotransfected with the PGC-1α 3′-untranslated region (3′-UTR) but not those with mutated 3′-UTR. miR-202 suppressed PGC-1α protein expression, with no influence on its messenger RNA expression. PGC-1α mediated VSMC phenotypic transition and was correlated with reactive oxygen species production. In conclusion, miR-202 affects VSMC phenotypic transition by targeting PGC-1α expression, providing a novel target for varicose vein therapy.     

Functional variants of hepatocyte growth factor identified in patients with adolescent idiopathic scoliosis

The genetic etiology of adolescent idiopathic scoliosis (AIS) remains obscure. Whole-genome sequencing was performed in four members of one family. Then, we performed a rigorous computational analysis to determine the deleterious effects of the identified variants. Furthermore, the structural differences between the native hepatocyte growth factor (HGF) protein and a protein encoded by an HGF variant containing one mutation (p.T596M) were analyzed using molecular dynamic stimulation. A novel heterozygous mutation (p.T596M) within the HGF gene was identified and found to cosegregate with scoliosis phenotypes in three affected family members. Subsequent modeling and structure-based analyses supported the theory that this mutation is functionally deleterious. Functional analyses demonstrated that the HGF p.T596 M mutation changed the ability of the HGF protein to be secreted and impaired migration and invasion in HEK293T cells. Furthermore, an HGF knockdown zebrafish model exhibited a curly tailed phenotype. Mutation in HGF is associated with an autosomal dominant pattern of inheritance of AIS. This finding increases our understanding of the genetic heterogeneity of AIS.     

MUC-1 aptamer targeted superparamagnetic iron oxide nanoparticles for magnetic resonance imaging of pancreatic cancer in vivo and in vitro experiment

This study aims to explore the ability of magnetic resonance imaging (MRI) in mucin 1 (MUC1) modified superparamagnetic iron oxide nanoparticle (SPION) targeting human pancreatic cancer (PC). The MUC1 target-directed probe was prepared through MUC1 conjugated to SPION using the chemical method to assess its physiochemical characteristics, including hydration diameter, surface charge, and magnetic resonance signal. The cytotoxicity of MUC1-USPION was verified by MTS assay. BxPC-3 was cultured with MUC1-USPION and SPION in different concentrations. The combined condition of the targeted probes and cells were observed through Prussian blue staining. The nude mice model of pancreatic cancer was established to investigate the application of the probe. MRI was performed to determine the intensity of the signal of the transplanted tumor, while immunohistochemistry and Western blot analysis were performed to detect the expression of MUC1 after taking the transplanted tumor specimen. The particle size of the prepared molecular probe was 63.5 ± 3.2 nm, and the surface charge was 10.2 mV. Furthermore, the probe solution could significantly reduce the MRI at T₂, and the magnetic resonance transverse relaxation rate (ΔR²) has a linear relationship with the concentration of iron in the solution. The cell viability of MUC1-USPION in different concentrations revealed no statistical difference, according to the MTS assay. In vitro, the MRI demonstrated decreased T2WI signal intensity in both groups, especially the targeting group. In vivo, MUC1 could selectively accumulate in the nude mice model, and significantly reduce the T₂ signal strength. In subsequent experiments, the expression of MUC1 was high in pancreatic cancer tissues, but low in normal pancreatic tissues, as determined by immunohistochemistry and Western blot analysis. The prepared samples can be combined with pancreatic cancer tissue specificity by in vivo imaging, providing reliable early in vivo imaging data for disease diagnosis.     

Effects of Na(+) channel and cell coupling abnormalities on vulnerability to reentry: a simulation study

The role of dynamic instabilities in the initiation of reentry in diseased (remodeled) hearts remains poorly explored. Using computer simulations, we studied the effects of altered Na(+) channel and cell coupling properties on the vulnerable window (VW) for reentry in simulated two-dimensional cardiac tissue with and without dynamic instabilities. We related the VW for reentry to effects on conduction velocity, action potential duration (APD), effective refractory period dispersion and restitution, and concordant and discordant APD alternans. We found the following: 1). reduced Na(+) current density and slowed recovery promoted postrepolarization refractoriness and enhanced concordant and discordant APD alternans, which increased the VW for reentry; 2). uniformly reduced cell coupling had little effect on cellular electrophysiological properties and the VW for reentry. However, randomly reduced cell coupling combined with decoupling promoted APD dispersion and alternans, which also increased the VW for reentry; 3). the combination of decreased Na(+) channel conductance, slowed Na(+) channel recovery, and cellular uncoupling synergistically increased the VW for reentry; and 4) the VW for reentry was greater when APD restitution slope was steep than when it was flat. In summary, altered Na(+) channel and cellular coupling properties increase vulnerability to reentrant arrhythmias. In remodeled hearts with altered Na(+) channel properties and cellular uncoupling, dynamic instabilities arising from electrical restitution exert important influences on the VW for reentry.     

The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.