Profiling analysis of long non-coding RNAs in early postnatal mouse hearts

Mammalian cardiomyocytes undergo a critical hyperplastic-to-hypertrophic growth transition at early postnatal age, which is important in establishing normal physiological function of postnatal hearts. In the current study, we intended to explore the role of long non-coding (lnc) RNAs in this transitional stage. We analyzed lncRNA expression profiles in mouse hearts at postnatal day (P) 1, P7 and P28 via microarray. We identified 1,146 differentially expressed lncRNAs with more than 2.0-fold change when compared the expression profiles of P1 to P7, P1 to P28, and P7 to P28. The neighboring genes of these differentially expressed lncRNAs were mainly involved in DNA replication-associated biological processes. We were particularly interested in one novel cardiac-enriched lncRNA, ENSMUST00000117266, whose expression was dramatically down-regulated from P1 to P28 and was also sensitive to hypoxia, paraquat, and myocardial infarction. Knockdown ENSMUST00000117266 led to a significant increase of neonatal mouse cardiomyocytes in G0/G1 phase and reduction in G2/M phase, suggesting that ENSMUST00000117266 is involved in regulating cardiomyocyte proliferative activity and is likely associated with hyperplastic-to-hypertrophic growth transition. In conclusion, our data have identified a large group of lncRNAs presented in the early postnatal mouse heart. Some of these lncRNAs may have important functions in cardiac hyperplastic-to-hypertrophic growth transition

Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/β-catenin pathway

Background In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level. Objective Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia. Methods and Results We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of β-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of β-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery. Conclusion These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/β-catenin-dependent manner

TRPA1 Promotes Cardiac Myofibroblast Transdifferentiation after Myocardial Infarction Injury via the Calcineurin-NFAT-DYRK1A Signaling Pathway

Cardiac fibroblasts (CFs) are a critical cell population responsible for myocardial extracellular matrix homeostasis. After stimulation by myocardial infarction (MI), CFs transdifferentiate into cardiac myofibroblasts (CMFs) and play a fundamental role in the fibrotic healing response. Transient receptor potential ankyrin 1 (TRPA1) channels are cationic ion channels with a high fractional Ca2+ current, and they are known to influence cardiac function after MI injury; however, the molecular mechanisms regulating CMF transdifferentiation remain poorly understood. TRPA1 knockout mice, their wild-type littermates, and mice pretreated with the TRPA1 agonist cinnamaldehyde (CA) were subjected to MI injury and monitored for survival, cardiac function, and fibrotic remodeling. TRPA1 can drive myofibroblast transdifferentiation initiated 1 week after MI injury. In addition, we explored the underlying mechanisms via in vitro experiments through gene transfection alone or in combination with inhibitor treatment. TRPA1 overexpression fully activated CMF transformation, while CFs lacking TRPA1 were refractory to transforming growth factor β- (TGF-β-) induced transdifferentiation. TGF-β enhanced TRPA1 expression, which promoted the Ca2+-responsive activation of calcineurin (CaN). Moreover, dual-specificity tyrosine-regulated kinase-1a (DYRK1A) regulated CaN-mediated NFAT nuclear translocation and TRPA1-dependent transdifferentiation. These findings suggest a potential therapeutic role for TRPA1 in the regulation of CMF transdifferentiation in response to MI injury and indicate a comprehensive pathway driving CMF formation in conjunction with TGF-β, Ca2+ influx, CaN, NFATc3, and DYRK1A

Post-Natal Inhibition of NF-κB Activation Prevents Renal Damage Caused by Prenatal LPS Exposure

<div><p>Prenatal exposure to an inflammatory stimulus has been shown to cause renal damage in offspring. Our present study explored the role of intra-renal NF-κB activation in the development of progressive renal fibrosis in offspring that underwent prenatal exposure to an inflammatory stimulus. Time-dated pregnant rats were treated with saline (control group) or 0.79 mg/kg lipopolysaccharide (LPS) through intra-peritoneal injection on gestational day 8, 10 and 12. At the age of 7 weeks, offspring from control or LPS group were treated with either tap water (Con+Ve or LPS+Ve group) or pyrollidine dithiocarbamate (PDTC, 120mg/L), a NF-κB inhibitor, <i>via</i> drinking water starting (Con+PDTC or LPS+PDTC group), respectively, till the age of 20 or 68 weeks. The gross structure of kidney was assessed by hematoxylin-eosin, periodic acid–Schiff staining and Sirius red staining. The expression levels of TNF-α, IL-6, α-smooth muscle actin (α-SMA) and renin-angiotensin system (RAS) genes were determined by real time polymerase chain reaction and/or immunohistochemical staining. Our data showed that post-natal persistent PDTC administration efficiently repressed intra-renal NF-κB activation, TNF-α and IL-6 expression. Post-natal PDTC also prevented intra-renal glycogen deposition and collagenous fiber generation as evident by the reduced expression of collagen III and interstitial α-SMA in offspring of prenatal LPS exposure. Furthermore, post-natal PDTC administration reversed the intra-renal renin-angiotensin system (RAS) over-activity in offspring of prenatal LPS exposure. In conclusion, prenatal inflammatory exposure results in offspring’s intra-renal NF-κB activation along with inflammation which cross-talked with excessive RAS activation that caused exacerbation of renal fibrosis and dysfunction in the offspring. Thus, early life prevention of NF-κB activation may be a potential preventive strategy for chronic renal inflammation and progressive renal damage.</p></div

NF-κB activation and increased expression of pro-inflammatory cytokines existed in the renal tissue of offspring that received prenatal LPS exposure.

<p>Relative protein expression of phospho-p65^ser536 and total p65 at the age of 20 weeks was determined by immunoblotting. Representative plots in each group and statistical data of relative densitometry, normalized by GAPDH, were shown (A). The mRNA expressions of <i>TNF-α</i> (B) and <i>IL-6</i> (C) in the kidney at the age of 20 weeks were determined by realtime RT-PCR. Data are presented as mean ± SD. n = 6 offspring per group for (A); n = 7 offspring in each group for (B) and (C). * and ** indicate P<0.05 and P<0.01, respectively, which denote statistical comparison between the two marked treatment groups (Two-way ANOVA for followed by Dunnett T3 test for inter-group comparison (A, B and C)). Con+Ve group, offspring rats from maternal saline treatment together with post-natal saline treatment; LPS+Ve group, offspring rats from maternal LPS exposure together with post-natal saline treatment; LPS+PDTC group, offspring rats from maternal LPS exposure together with post-natal PDTC treatment; Con+PDTC group, offspring rats from maternal saline treatment together post-natal PDTC treatment.</p

Increased protein level of intrarenal α-SMA expression in offspring of prenatal LPS exposure could be reversed by post-natal NF-κB inhibition.

<p>Renal α-SMA expression in offspring at the age of 68 weeks was determined by immunohistochemistry (A) and semi-quantitation of its positive area and density was show in (B). Data are presented as mean ± SD. n = 7 offspring and 4–5 pictures from each offspring were quantified for (B). ** indicates P<0.01, which denotes statistical comparison between the two marked treatment groups (Two-way ANOVA followed by LSD test (B) for inter-group comparison). Indications of Con+Ve, LPS+Ve, LPS+PDTC and Con+PDTC are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153434#pone.0153434.g001" target="_blank">Fig 1</a>.</p

Renal collagen deposition in offspring of prenatal LPS exposure could be reversed by post-natal NF-κB inhibition.

<p>(A) Level of renal collagen deposition in 68-week-old offspring was assessed by Sirius red staining and observed under both white microscope (top panel) and polarized microscope (Arrow indicates collagen III accumulation, bottom panel). Semi-quantitation of relative Sirius red-positive area was shown (B). Relative mRNA expressions of <i>collagen type1</i> (<i>Col1a1</i>) and <i>collagen type 3</i> (<i>Col3a1</i>) (C) were determined by realtime RT-PCR in kidney at the age of 20 weeks. Data are presented as mean ± SD. n = 7 offspring and 4–5 pictures from each offspring were quantified for (B). n = 7 offspring in each group for (C). * and ** indicate P<0.05 and P<0.01, respectively, which denote statistical comparison between the two marked treatment groups (Two-way ANOVA followed by Dunnett T3 test (B) or LSD test (C) for inter-group comparison). Indications of Con+Ve, LPS+Ve, LPS+PDTC and Con+PDTC are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153434#pone.0153434.g001" target="_blank">Fig 1</a>.</p