Canonical pathways for validating steroid-associated osteonecrosis in mice.

The present study aimed to establish and evaluate a preclinical model of steroid-associated osteonecrosis (SAON) in mice. Sixteen 24-week-old male C57BL/6 mice were used to establish SAON by two intraperitoneal injections of lipopolysaccharide (LPS), followed by three subcutaneous injections of methylprednisolone (MPS). Each injection was conducted on working day, with an interval of 24 h. Six cycles of injections were conducted. Additional twelve mice (age- and gender-matched) were used as normal controls. At 2 and 6 weeks after completing induction, bilateral femora and bilateral tibiae were collected for histological examination, micro-CT scanning, and bulk RNA sequencing. All mice were alive until sacrificed at the indicated time points. The typical SAON lesion was identified by histological evaluation at week 2 and week 6 with increased lacunae and TUNEL+ osteocytes. Micro-CT showed significant bone degeneration at week 6 in SAON model. Histology and histomorphometry showed significantly lower Runx2+ area, mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR/BS), type H vessels, Ki67+ (proliferating) cells, and higher marrow fat fraction, osteoclast number and TNFα+ areas in SAON group. Bulk RNA-seq revealed changed canonical signaling pathways regulating cell cycle, angiogenesis, osteogenesis, and osteoclastogenesis in the SAON group. The present study successfully established SAON in mice with a combination treatment of LPS and MPS, which could be considered a reliable and reproducible animal model to study the pathophysiology and molecular mechanism of early-stage SAON and to develop potential therapeutic approaches for the prevention and treatment of SAON.

Decreased PKG transcription mediated by PI3K/Akt/FoxO1 pathway is involved in the development of nitroglycerin tolerance.

Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10-5 M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg-1, tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of PI3K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1.

Homocysteine ameliorates the endothelium-independent hypoxic vasoconstriction via the suppression of phosphatidylinositol 3-kinase/Akt pathway in porcine coronary arteries.

OBJECTIVE: Endothelium-independent coronary vasoconstriction induced by continuous hypoxia contributes to the development of ischemic heart diseases. Acute elevation of homocysteine (Hcy) has a potent of vasodilation. The present study aims to investigate the role of Hcy in endothelium-independent hypoxic coronary vasoconstriction and its underlying mechanisms. METHODS AND RESULTS: Vessel tension of isolated porcine coronary arteries was measured by organ chamber study and the protein expression were detected by western blot. A sustained contraction of porcine coronary artery was induced when exposed to prolonged hypoxia for more than 15 min, which was significantly reduced by Hcy in a dose-dependent manner but not affected by cysteine or N-acetyl-l-cysteine. Phosphorylated myosin light chain (MLC-p) at Ser19 was decreased when exposure to hypoxia for 15 min, and could be reversed by prolonged hypoxia for 30 and 60 min. The recovery of MLC-p at Ser19 by hypoxia for more than 30 min could be abolished by Hcy. The protein levels of phosphorylated Akt at Ser473 and phosphorylated P85 at Tyr508 were decreased by Hcy in normoxia, and were also reduced exposure to hypoxia for 15 min and then augmented by prolonged hypoxia for more than 30 min, which could be prevented by Hcy. The protein level of P110α was not affected by Hcy or prolonged hypoxia. CONCLUSIONS: This study demonstrates that Hcy can ameliorate the endothelium-independent hypoxic coronary vasoconstriction, in which the inhibition of PI3K/Akt signaling pathway may be involved.

Plasma endothelin-1-related peptides as the prognostic biomarkers for heart failure: A PRISMA-compliant meta-analysis.

BACKGROUND: Most studies reported that high plasma endothelin-1 (ET-1), big ET-1, and C-terminal proET-1 (CT-proET-1) were correlated with poor prognosis of heart failure (HF). However, available evidence remains controversial. To help solve the debate, we collected all the available studies and performed a meta-analysis. METHODS: We searched the databases covering Embase, PubMed, Ovid, and Web of Science on June 28, 2017. The hazard ratio (HR) or risk ratio (RR) and its 95% confidence intervals (CIs) were collected and calculated by use of a random-effect model. Heterogeneity was assessed by Cochran's Q test, and publication bias was assessed by funnel plots with Egger's and Begg's linear regression test. RESULTS: Thirty-two studies with 18,497 patients were included in the analysis. Results showed that circulating ET-1, big ET-1, and CT-proET-1 were positively correlated with high risk of adverse outcomes, with pooled RRs (95% CIs) of 2.22 (1.82-2.71, P < .001), 2.47 (1.93-3.17, P < .001), and 2.27 (1.57-3.29, P < .001), respectively. In the subgroup of death as primary outcome, the pooled RRs (95% CIs) were 2.13 (1.68-2.70, P < .001), 2.55 (1.82-3.57, P < .001), and 2.02 (1.39-2.92, P < .001) for ET-1, big ET-1, and CT-proET-1, respectively. No significant publication bias was observed in this study. CONCLUSION: Our meta-analysis provided evidence that increased plasma levels of ET-1, big ET-1, and CT-proET-1 were associated with poor prognosis or mortality for HF populations.

CTRP3 promotes energy production by inducing mitochondrial ROS and up-expression of PGC-1α in vascular smooth muscle cells.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine with modulation effects on metabolism and inflammation. Adenosine triphosphate (ATP) exerts multiple biological effects in vascular smooth muscle cells (VSMCs) and energy imbalance is involved in vascular diseases. This study aimed to explore the effect of CTRP3 on energy production and its underlying mechanism in VSMCs. Our results indicated that exogenous CTRP3 increased ATP synthesis and the protein expression of oxidative phosphorylation (OXPHOS)-related molecules, including peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, sirtuin-3 (SIRT3), complex I, II, III, and V in cultured VSMCs. Depletion of endogenous CTRP3 by small interfering RNA (siRNA) reduced ATP synthesis and the expression of those molecules. PGC-1α knockdown abrogated CTRP3-induced ATP production and OXPHOS-related protein expression. Furthermore, CTRP3 increased mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential level. Pretreatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, and cyanidem-chlorophenylhydrazone, an uncoupler of OXPHOS, suppressed CTRP3-induced ROS production, PGC-1α expression and ATP synthesis. In conclusion, CTRP3 modulates mitochondrial energy production through targets of ROS and PGC-1α in VSMCs.

Endothelium-Independent Hypoxic Contraction Is Prevented Specifically by Nitroglycerin via Inhibition of Akt Kinase in Porcine Coronary Artery.

Objective. Hypoxia-induced sustained contraction of porcine coronary artery is endothelium-independent and mediated by PI3K/Akt/Rho kinase. Nitroglycerin (NTG) is a vasodilator used to treat angina pectoris and acute heart failure. The present study was to determine the role of NTG in hypoxia-induced endothelium-independent contraction and the underlying mechanism. Methods and Results. Organ chamber technique was used to measure the isometric vessel tension of isolated porcine coronary arteries. Protein levels of phosphorylated and total Akt were determined by western blot. A sustained contraction of porcine coronary arteries induced by hypoxia was significantly reduced by NTG but not by isoproterenol. This contraction was also inhibited by DETA NONOate, 8-Br-cGMP, which can be reversed by ODQ, and Rp-8-Br-PET-cGMPS. The restored contraction was blocked by LY294002. The reduction of Akt-p at Ser-473 by NTG, DETA NONOate, and 8-Br-cGMP was significantly inhibited by ODQ, PKG-I. The decrease in Akt-p level by NTG and 8-Br-cGMP was prevented by calyculin A but not by okadaic acid. Conclusions. These results demonstrated that the endothelium-independent sustained hypoxic vasoconstriction can be prevented by NTG and that the inhibition of PI3K/Akt signaling pathway may be involved.