Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis

<div><p>Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.</p></div

MiR-26a mediates the SMC phenotype regulation co-cultured with miR-206-overexpressing HUVECs.

<p>(A) Real-time PCR analysis of SRF expression in SMCs. (B) Western blot analysis of SRF protein level in SMCs. (C, D) Real-time PCR analysis of phenotype-related miRNA expression in SMCs co-cultured with miR-206-overexpressing HUVECs (C) and antagomiR-206-overexpressing HUVECs (D). (E) Real-time PCR analysis of miR-26a expression in SMCs treated with exosomes from miR-206-overexpressing HUVECs. (F) Real-time PCR analysis of miR-26a expression in the total exosomes from the same amounts of mock-, miR-206, antagomiR-206, siARF6 and siNCX1-transduced HUVECs. (G, H) Real-time PCR analysis of miR-26a expression (G) and phenotype marker gene expression (H) in SMCs co-cultured with HUVECs co-transfected with antagomiR-26a and antagomiR-206 or mock. Three independent experiments were performed for each condition, and data are presented as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01 versus control group.</p

HUVEC exosomes attenuate the SMC contractile phenotype.

<p>(A) GFP expression in SMCs visualized by confocal microscopy at 600× magnification. (B) Western blot analysis of GFP protein in SMCs. (C) Real-time PCR analysis of cel-miR-39 expression in SMCs. (D) Determination of exosome size using a Zetasizer Nano instrument and transmission electron microscopy (indicated by black arrow). (E) Real-time PCR analysis of contractile marker gene expression (α-SMA, Smoothelin and Calponin) in SMCs in the presence or absence of exosomes. (F) Western blot analysis of contractile marker gene expression (α-SMA and Calponin) in SMCs in the presence or absence of exosomes. (G) Confocal microscopy of intracellular actin filament organization of SMCs by staining with Phalloidin-TRITC at 600× magnification. (H) Real-time PCR analysis of phenotype marker gene expression (α-SMA, Smoothelin and Calponin) in SMCs treated with exosomes from miR-206-overexpressing HUVECs. Three independent experiments were performed for each condition, and the data are presented as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01 versus control group.</p

Ectopic miR-206 expression in HUVECs induces the SMC contractile phenotype.

<p>(A) An <i>in vitro</i> transwell system was used for co-culture experiments. HUVECs were seeded in the top compartment and SMCs were cultured in the bottom compartment. Exosomes mediated the regulation of SMCs by HUVECs. (B) Real-time PCR analysis of contractile marker gene expression (α-SMA, Smoothelin and Calponin) in SMCs co-cultured with miR-206-overexpressing HUVECs. (C) Real-time PCR analysis of contractile marker gene expression (α-SMA, Smoothelin and Calponin) in SMCs co-cultured with antagomiR-206-overexpressing HUVECs. (D) Western blot analysis of contractile marker gene expression (α-SMA and Calponin) in SMCs co-cultured with miR-206-overexpressing HUVECs. Three independent experiments were performed for each condition, and data are presented as the mean ± SEM. * <i>p</i><0.05 and ** <i>p</i><0.01 versus control group.</p

FeCl₃‑Catalyzed Stereoselective Construction of Spirooxindole Tetrahydroquinolines via Tandem 1,5-Hydride Transfer/Ring Closure

An efficient FeCl₃-catalyzed stereoselective intramolecular tandem 1,5-hydride transfer/ring closure reaction was developed. The method allows for the formation of structurally diverse spirooxindole tetrahydroquinolines in high yields (up to 98%) with good to excellent levels of diastereoselectivity (up to 99:1 dr). The catalytic enantioselective variant of this process was also investigated preliminarily with a chiral BINOL-derived phosphoric acid

siRNA Sequences.

<p>siRNA Sequences.</p

Real-time PCR primers.

<p>Real-time PCR primers.</p

ARF6 and NCX1 mediate exosome production in HUVECs.

<p>(A) DHPE staining of intracellular MVBs assayed by confocal microscopy at 600× magnification. (B) Western blot analysis of Hsp70 protein expression in exosomes secreted from ARF6 knockdown HUVECs. (C) Fluo-3AM staining of intracellular calcium assayed by confocal microscopy at 600× magnification. (D) Western blot analysis of Hsp70 protein expression in exosomes secreted from NCX1 knockdown HUVECs.</p

FeCl₃‑Catalyzed Stereoselective Construction of Spirooxindole Tetrahydroquinolines via Tandem 1,5-Hydride Transfer/Ring Closure

An efficient FeCl₃-catalyzed stereoselective intramolecular tandem 1,5-hydride transfer/ring closure reaction was developed. The method allows for the formation of structurally diverse spirooxindole tetrahydroquinolines in high yields (up to 98%) with good to excellent levels of diastereoselectivity (up to 99:1 dr). The catalytic enantioselective variant of this process was also investigated preliminarily with a chiral BINOL-derived phosphoric acid

Fragmentation Study of Limonoids from <i>Turraae pubescens</i> by Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometry

<p>Nine representative limonoids isolated from <i>Turraae pubescens</i> were investigated by electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. Although the structures of these compounds are similar, the corresponding fragmentation patterns and mass spectrometry and tandem mass spectrometry (MS/MS) spectra are clearly different. For Turrapubin A–C, product ions can be detected in both low and high mass ranges. A McLafferty-type rearrangement is the only way for the cleavage of C9–C10. For 11-epi-toonacilin, Turrapubin E, Turraflorin A, 11-epi-23-hydroxytoonacilide, Turrapubin H and 11-epi-21-hydroxytoonacilide, the cleavage of C9–C10 goes through two different ways, including McLafferty-type rearrangement and homolytic cleavage. The relative abundances of product ions from McLafferty-type rearrangement for 11-epi-toonacilin, 11-epi-23-hydroxytoonacilide, and Turrapubin H are high, while those for Turrapubin E, Turraflorin A, and 11-epi-21-hydroxytoonacilide are low. A pair of epimers was distinguished unambiguously by MS/MS spectra. It was found that the substituent group at C-1, hydroxy group, O atom linked to C-14 and C-15, and the oxygenated furan ring were the important factors leading to the differences of their MS/MS spectra.</p