NFAT5 promotes arteriogenesis via MCP-1-dependent monocyte recruitment.

Studies have demonstrated that nuclear factor of activated T cells 5 (NFAT5) is not only a tonicity-responsive transcription factor but also activated by other stimuli, so we aim to investigate whether NFAT5 participates in collateral arteries formation in rats. We performed femoral artery ligature (FAL) in rats for hindlimb ischaemia model and found that NFAT5 was up-regulated in rat adductors with FAL compared with sham group. Knockdown of NFAT5 with locally injection of adenovirus-mediated NFAT5-shRNA in rats significantly inhibited hindlimb blood perfusion recovery and arteriogenesis. Moreover, NFAT5 knockdown decreased macrophages infiltration and monocyte chemotactic protein-1 (MCP-1) expression in rats adductors. In vitro, with interleukin-1ß (IL-1ß) stimulation and loss-of-function studies, we demonstrated that NFAT5 knockdown inhibits MCP-1 expression in endothelial cells and chemotaxis of THP-1 cells regulated by ERK1/2 pathway. More importantly, exogenous MCP-1 delivery could recover hindlimb blood perfusion, promote arteriogenesis and macrophages infiltration in rats after FAL, which were depressed by NFAT5 knockdown. Besides, NFAT5 knockdown also inhibited angiogenesis in gastrocnemius muscles in rats. Our results indicate that NFAT5 is a critical regulator of arteriogenesis and angiogenesis via MCP-1-dependent monocyte recruitment, suggesting that NFAT5 may represent an alternative therapeutic target for ischaemic diseases.

Induction of microRNA-199 by Nitric Oxide in Endothelial Cells Is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase.

BACKGROUND: Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, because of the dysfunction of prostaglandin I2 synthase (PTGIS). MicroRNAs repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression. METHODS: Nitrovasodilator resistance was induced by nitroglycerin (100 mg·kg-1·d-1, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide donors were assessed in an organ chamber. The expression levels of microRNA-199 (miR-199)a/b were assayed by quantitative reverse transcription polymerase chain reaction or fluorescent in situ hybridization. RESULTS: In cultured human umbilical vein endothelial cells, nitric oxide donors induced miR-199a/b endogenous expression and downregulated PTGIS gene expression, both of which were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976 to 982 in the 3'-untranslated region of PTGIS mRNA is a target of miR-199a/b. Gain functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and human umbilical vein endothelial cells. Furthermore, nitroglycerin-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in human umbilical vein endothelial cells. In Apoe-/- mice, nitroglycerin induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. It is important that the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to nitroglycerin/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of the PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to nitroglycerin infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in nitroglycerin-infused Apoe-/- mice, whereas U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance. CONCLUSIONS: Nitric oxide-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.

miRNA expression pattern associated with prognosis in elderly patients with advanced OPSC and OCC.

The long-term survival for elderly patients with advanced ovarian papillary serous carcinoma (OPSC) does not exceed 30%, and the incidence and prognosis rise continuously after menopause. The aim of this study was to identify the differences in key miRNAs and their potential regulators through miRNA microarray analysis, functional target prediction, and clinical outcome between the elderly patients with advanced OPSC and ovarian clear cell carcinoma (OCC) who all suffered poor prognosis, to identify the pathogenetic basis, and to improve the understanding of the molecular basis of advanced OPCS in elderly patients. Through microarray analysis, we found 52 unique miRNAs with significant fold­change in expression levels, of which 9 were upregulated, whereas 43 were downregulated in OCC patients compared to elderly OPSC patients with advanced stage. Among these prediction miRNAs, miR-30a, miR-30e and miR-505 were found to have some common cancer-related targets. Lower expression of these three miRNAs of advanced OPSC in elderly patients, all associated with significantly poorer survival rate, strongly suggesting that they could be critical oncogenes and take important roles in OPSC etiology in elderly patients with advantaged stage. Functional analyses support the above hypothesis. Their targets, ATF3, STMN1 and MYC suggest that OPSC also has aggressive biological behavior when presented with advanced stage, and support the epidemiology results that incidence and mortality of advanced OPSC increases continuously. miR-30a, miR-30e and miR-505 may be important pathogenetic factors for OPSC in elderly patients with advanced stage. Age could be regarded as a continuous covariate in this process. This may improve the understanding of molecular underpinnings of advanced OPSC in elderly patients, and provide improved diagnostic, prognostic and therapeutic approaches.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.