Background: Myocardial fibrosis is a common pathophysiological change in cardiovascular disease, which can cause cardiac dysfunction and even sudden death. Excessively activated fibroblasts proliferate and secret excessive extracellular matrix (ECM) components, resulting in normal cardiac structural damage and cardiac fibrosis. We previously found that human endothelial progenitor cell (EPC)-derived exosomes, after hypoxia/reoxygenation (H/R) induction, could significantly increase the mesenchymal-endothelial transition (MEndoT) compared to normal culture EPC-derived exosomes. Exosomes have been shown to carry different nucleic acids, including microRNAs. However, the effects of microRNAs in EPC-derived exosomes on MEndoT and myocardial fibrosis remain unknown. Methods: EPCs were isolated from human peripheral blood, and fibroblasts were isolated from rat hearts, then transfected with miR-133 inhibitor, si-YBX-1, and ov-YBX-1 into EPCs. After H/R induction for 48 h, isolation and characterization of exosomes derived from human EPCs were performed. Finally, fibroblasts were treated by exosome at 48 h. The expression of miR-133 was measured by qRT-PCR; YBX-1 expression was measured by qRT-PCR and western blot. Angiopoiesis was measured by tube formation assay. Endothelial markers and fibrosis markers were measured by western blot. Results: H/R treatment promoted miR-133 expression in EPCs and EPC-derived exosomes. miR-133 could be incorporated into exosomes and transmitted to cardiac fibroblasts, increasing the angiogenesis and MEndoT of cardiac fibroblasts. miR-133 silencing in H/R-induced EPCs could inhibit miR-133 expression in EPCs and EPCsderived exosomes. miR-133 silencing in H/R-induced EPCs could inhibit the angiogenesis and MEndoT of cardiac fibroblasts and reverse the effect of H/R treatment. Additionally, miR-133 was specially sorted into H/R-induced EPCderived exosomes via YBX-1. YBX-1 silencing inhibited miR-133 transfer and reduced fibroblast angiogenesis and MEndoT. Conclusion: miR-133 was specially sorted into H/R-induced EPC-derived exosomes via YBX-1 to increase fibroblast angiogenesis and MEndoT.

Dysfunction of endothelial cells plays a key role in the pathogenesis of diabetic atherosclerosis. High glucose (HG) has been found as a key factor in the progression of diabetic complications, including atherosclerosis. PI3K/Akt/eNOS signaling pathway has been shown to involve in HG-induced vascular injuries. Hydrogen sulfide (H 2 S) has been found to exhibit protective effects on HG-induced vascular injuries. Moreover, H 2 S activates PI3K/Akt/eNOS pathway in endothelial cells. Thus, the present study aimed to determine if H 2 S exerts protective effects against HG-induced injuries of human umbilical vein endothelial cells (HUVECs) via activating PI3K/Akt/eNOS signaling. Materials and Methods: The endothelial protective effects of H 2 S were evaluated and compared to the controlled groups. Cell viability, cell migration and tube formation were determined by in vitro functional assays; protein levels were evaluated by Western blot assay and ELISA; cell apoptosis was determined by Hoechst 33258 nuclear staining; Reactive oxygen species (ROS) production was evaluated by the ROS detection kit. Results: HG treatment significantly inhibited PI3K/Akt/eNOS signaling in HUVECs, which was partially reversed by the H2S treatment. HG treatment inhibited cell viability of HUVECs, which were markedly prevented by H 2 S or PI3K agonist Y-P 740. HG treatment also induced HUVEC cell apoptosis by increasing the protein levels of cleaved caspase 3, Bax and Bcl-2, which were significantly attenuated by H 2 S or 740 Y-P. ROS production and gp91 phox protein level were increased by HG treatment in HUVECs and this effect can be blocked by the treatment with H 2 S or Y-P 740. Moreover, HG treatment increased the protein levels of pro-inflammatory cytokines, caspase-1 and phosphorylated JNK, which was significantly attenuated by H 2 S or Y-P 740. Importantly, the cytoprotective effect of H 2 S against HG-induced injury was inhibited by LY294002 (an inhibitor of PI3K/Akt/eNOS signaling pathway). Conclusion: The present study demonstrated that exogenous H 2 S protects endothelial cells against HG-induced injuries by activating PI3K/Akt/eNOS pathway. Based on the above findings, we proposed that reduced endogenous H 2 S levels and the subsequent PI3K/Akt/ eNOS signaling impairment may be the important pathophysiological mechanism underlying hyperglycemia-induced vascular injuries.

Myocardial ischemia/reperfusion (I/R) injury is a clinically severe complication, which can cause high rates of disability and mortality particularly in patients with myocardial infarction, yet the molecular mechanisms underlying this process remain unclear. This study aimed to explore the protective effects of β-sitosterol against myocardial I/R injury and to elucidate the underlying molecular mechanisms. Our results showed that hypoxia/reoxygenation (H/R) treatment suppressed cell viability, induced cell apoptosis and reactive oxygen species production, increased caspase-3 and -9 activities, upregulated caspase-3 and -9 protein expressions, downregulated the Bcl-2 protein expression, and reduced the mitochondrial membrane potential. β-Sitosterol treatment attenuated H/R-induced cardiomyocyte injury. Moreover, β-sitosterol treatment counteracted the inhibitory effects of H/R treatment on the peroxisome proliferator-activated receptor gamma (PPARγ) expression and enhanced effects of H/R treatment on the NF-κB expression in cardiomyocytes. Furthermore, inhibition of PPARγ impaired the protective actions of β-sitosterol against H/R-induced cardiomyocyte injury. In the I/R rats, β-sitosterol treatment reduced the myocardial infarcted size and apoptosis, which was attenuated by the inhibition of PPARγ. In conclusion, our results demonstrate that β-sitosterol protected against in vitro H/R-induced cardiomyocyte injury and in vivo myocardial I/R injury. The β-sitosterol-mediated cardioprotective effects may involve the modulation of PPARγ/NF-κB signalling during myocardial I/R injury. Further studies are required to further explore the clinical application of β-sitosterol in the myocardial I/R injury.

Objectives This study aimed to evaluate berberine (BBR) effects on myocardial hypertrophy (MH) and associated mechanisms. Methods BBR effects on MH were evaluated in rats with constriction of abdominal aorta (CAA). qRT-PCR assay was used to measure MH-related genes, long non-coding RNAs (lncRNAs) and autophagy-related genes expressions. Western blot was performed to detect autophagy markers expression. Filamentous actin and phalloidin expressions were detected using immunofluorescence assay. Key findings BBR significantly attenuated CAA-induced MH and cardiomyocyte enlargement. CAA upregulated b myosin heavy chain and atrial natriuretic peptide expressions in heart tissues, which was attenuated by BBR. BBR suppressed myocardial infarction associated transcript (MIAT) expression in rats with CAA. p62 mRNA expression was upregulated and beclin1 and autophagy related 5 were downregulated in CAA versus control groups. The effects were abolished by BBR.In vitro studies showed that BBR ameliorated angiotensin II-induced MH and attenuated Ang II-induced MIAT expression in H9C2 cells. Expressions of phosphorylated mTOR, phosphorylated AMPK and LC3 were upregulated in H9C2 cells after Ang II stimulation, and the effects were abolished by BBR. Conclusions BBR exerted beneficial effects on MH induced by CCA, and the mechanisms were associated with decreased MIAT expression and enhanced autophagy.

Berberine (BBR) is an effective component of Huanglian and has shown to attenuate atherosclerosis (AS); however, the detailed mechanism of BBR-mediated protective actions against AS remains elusive. This study was undertaken to examine the effects of BBR on aortic atherosclerotic plaque stability and the expression of autophagy-related proteins in AS rats with damp-heat syndrome or yang deficiency. Methods: Thirty SD rats were randomly divided into (1) control (CON); (2) damp-heat syndrome atherosclerosis (AS + DH); (3) yang deficiency syndrome atherosclerosis (AS + YX); (4) dampheat syndrome atherosclerosis + BBR (AS + DH + BBR); (5) yang deficiency syndrome, atherosclerosis + BBR (AS + YX + BBR); and (6) damp-heat syndrome, atherosclerosis + BBR + 3-methyladenine (AS + DH + BBR + 3-MA) (n = 5/group) groups. Pathological morphology, macrophage plaque infiltration, inflammation, and LC3-II and P62 expression were assessed. Results: Compared with the CON group, the AS + DH and AS + YX groups had an increased plaque area in the aortic tissue with substantial foam cell and macrophage infiltration, and increased levels of IL-1β and TNF-α (P < 0.01). After four weeks of BBR intervention, the plaque area in the AS + DH + BBR group was reduced with decreased foam cells and macrophage infiltration, and decreased levels of TNF-α and IL-1β, whereas LC3-II protein expression was increased and P62 protein expression was decreased in the AS + DH + BBR group when compared to AS + DH group. In addition, the AS + DH + BBR + 3-MA group exhibited a significantly enlarged plaque, substantial foam cell and macrophage infiltration, increased levels of IL-1β and TNF-α, and decreased LC3-II and P62 (P < 0.01) expression when compared to the AS + DH + BBR group. Conclusion: Our results indicated that the BBR could inhibit arterial plaque formation and alleviate the inflammatory response in the aortic tissues in the AS rats with damp-heat syndrome possibly via promoting autophagy. The molecular mechanisms of BBR-mediated protective effects in this animal model still require further investigation.