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Background: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis after angioplasty or stent placement. VSMCs reprogram their metabolism to meet the increased requirements of lipids, proteins, and nucleotides for their proliferation. De novo purine synthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined. Methods: De novo purine synthesis in proliferative VSMCs was evaluated by liquid chromatography-tandem mass spectrometry. The expression of ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last 2 steps of the de novo purine synthesis pathway, was assessed in VSMCs of proliferative arterial neointima. Global and VSMC-specific knockout of Atic mice were generated and used for examining the role of ATIC-associated purine metabolism in the formation of arterial neointima and atherosclerotic lesions. Results: In this study, we found that de novo purine synthesis was increased in proliferative VSMCs. Upregulated purine synthesis genes, including ATIC, were observed in the neointima of the injured vessels and atherosclerotic lesions both in mice and humans. Global or specific knockout of Atic in VSMCs inhibited cell proliferation, attenuating the arterial neointima in models of mouse atherosclerosis and arterial restenosis. Conclusions: These results reveal that de novo purine synthesis plays an important role in VSMC proliferation in arterial disease. These findings suggest that targeting ATIC is a promising therapeutic approach to combat arterial diseases.

The mammalian epididymis provides an optimal and antioxidative fluid microenvironment for post-testicular sperm maturation by secretion of antioxidant scavengers and removal of excessive ROS. MicroRNAs (miRNAs) are expressed in the epididymis and involved in the regulation of epididymis physiology and functions. However, whether miRNAs are involved in regulating the antioxidant capacity and oxidative damage in the epididymis is not well understood. This study was designed to investigate the role of miR-542-3p in the regulation of antioxidant capacity and oxidative damage in the epididymis of goats. Firstly, we determined the expression of miR-542-3p and glutathione peroxidase 5 (GPx5) in the epididymis of young and adult goats using RT-qPCR assay, and found that miR-542-3p and GPx5 exhibited inverse expression levels. Our results showed that the expression level of miR-542-3p in epididymis was upregulated (P 0.05) in young goats compared to adult goats, whereas the expression level of GPx5 in epididymis was downregulated (P 0.01) in young goats compared to adult goats. Next, we further investigated the roles and potential mechanisms of miR-542-3p in epididymis using goat caput epididymal epithelial cells (GCEECs) isolated from Tai-hang goats (9-month-old). Our results showed that the overexpression of miR-542-3p in GCEECs transfected with miR-542-3p mimics resulted in decreased (P 0.05) antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT). Similarly, the overexpression of miR-542-3p in GCEECs resulted in decreased (P 0.05) glutathione (GSH) content and total antioxidant capacity (TAOC). In addition, the overexpression of miR-542-3p in GCEECs resulted in increased (P 0.05) malonaldehyde (MDA) content. The inverse results of SOD, CAT, GSH, TAOC and MDA were observed in the down-expression of miR-542-3p in GCEECs transfected with miR-542-3p inhibitors (P 0.05). Furthermore, GPx5 was confirmed to be a validated target of miR-542-3p in GCEECs using a dual-luciferase reporter assay, and transfection of miR-542-3p mimics decreased (P 0.05) the mRNA expression and protein level of GPx5. In conclusion, our results indicated that miR-542-3p reduced antioxidant capacity and increased oxidative damage in GCEECs by targeting GPx5. The present study further understands the regulation of antioxidant capacity and epididymal-specific GPx5 secretion in caput epididymidis.

Background: Transcriptional reconfiguration is central to heart failure, the most common cause of which is dilated cardiomyopathy (DCM). The effect of 3-dimensional chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. Methods: We generated a compendium of 3-dimensional epigenome and transcriptome maps from 101 biobanked human DCM and nonfailing heart tissues through highly integrative chromatin immunoprecipitation (H3K27ac [acetylation of lysine 27 on histone H3]), in situ high–throughput chromosome conformation capture, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin using sequencing, and RNA sequencing. We used human induced pluripotent stem cell–derived cardiomyocytes and mouse models to interrogate the key transcription factor implicated in 3-dimensional chromatin organization and transcriptional regulation in DCM pathogenesis. Results: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated in DCM development. For example, we identified that nontranscribing NPPA-AS1 (natriuretic peptide A antisense RNA 1) promoter functions as an enhancer and physically interacts with the NPPA (natriuretic peptide A) and NPPB (natriuretic peptide B) promoters, leading to the cotranscription of NPPA and NPPB in DCM hearts. We revealed that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (compartments, topologically associating domains) and their anchors unexpectedly had equivalent chromatin accessibility. We discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for HAND1 (heart and neural crest derivatives expressed 1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer–promoter connectome in DCM hearts, we performed comprehensive 3-dimensional epigenome mappings in human induced pluripotent stem cell–derived cardiomyocytes. We found that forced overexpression of HAND1 in human induced pluripotent stem cell–derived cardiomyocytes induced a distinct gain of enhancer–promoter connectivity and correspondingly increased the expression of their connected genes implicated in DCM pathogenesis, thus recapitulating the transcriptional signature in human DCM hearts. Electrophysiology analysis demonstrated that forced overexpression of HAND1 in human induced pluripotent stem cell–derived cardiomyocytes induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in dilated cardiac remodeling with impaired contractility/Ca 2+ handling in cardiomyocytes, increased ratio of heart weight/body weight, and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. Conclusions: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer–promoter connectome to drive DCM pathogenesis.

The objective of this study was to investigate the characteristics of ruminal microbial communities of alpacas (Lama pacos) and sheep (Ovis aries) fed three diets with varying ratios of roughage (corn stalk) to concentrate, 3:7 (LS), 5:5 (MS) and 7:3 (HS). Six alpacas (one-year-old and weighing 29.5 ± 7.1 kg) and six sheep (one-year-old and weighing 27.9 ± 2.7 kg) were used in this study, in a replicated 3 × 3 Latin square experiment. Total protozoa concentration was determined under the microscope; total fungi and methanogens were assessed using quantitative polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies; bacterial communities were investigated by targeted 16S rRNA gene (V3-V4 region) sequencing. The percentage of fungi was significantly higher in alpacas than in sheep under the LS diet, while the concentration of protozoa was significantly lower in alpacas under HS, MS and LS diets. The alpha diversity including Shannon, Chao l and ACE indices of bacterial communities was higher in alpacas than in sheep, under the LS diet. A total of 299 genera belonging to 22 phyla were observed in the forestomach of alpaca and sheep, with Bacteroidetes and Firmicutes dominating both animal species. Phyla Armatimonadetes and Fusobacteria, as well as 64 genera, were detected only in alpacas, whereas phyla Acidobacteria and Nitrospira, as well as 44 genera, were found only in sheep. The abundance of cellulolytic bacteria, including Butyrivibrio and Pseudobutyrivibrio, was higher in alpacas than in sheep under all three diets. These differences in the forestomach microbial communities partly explained why alpacas displayed a higher poor-quality roughage digestibility, and a lower methane production. Results also revealed that the adverse effects of high-concentrate diets (70%) were lesser in alpacas than in sheep.

Long noncoding RNAs (lncRNAs) play important roles in the development of vascular diseases. However, the effect of lncRNA NORAD on atherosclerosis remains unknown. This study aimed to investigate the effect NORAD on endothelial cell injury and atherosclerosis. Ox-LDL-treated human umbilical vein endothelial cells (HUVECs) and high-fat-diet (HFD)-fed ApoE−/− mice were used as in vitro and in vivo models. Results showed that NORAD-knockdown induced cell cycle arrest in G0/G1 phase, aggravated ox-LDL-induced cell viability reduction, cell apoptosis, and cell senescence along with the increased expression of Bax, P53, P21 and cleaved caspase-3 and the decreased expression of Bcl-2. The effect of NORAD on cell viability was further verified via NORAD-overexpression. NORAD- knockdown increased ox-LDL-induced reactive oxygen species, malondialdehyde, p-IKBα expression levels and NF-κB nuclear translocation. Proinflammatory molecules ICAM, VCAM, and IL-8 were also increased by NORAD- knockdown. Additionally, we identified the strong interaction of NORAD and IL-8 transcription repressor SFPQ in HUVECs. In ApoE−/− mice, NORAD-knockdown increased the lipid disorder and atherosclerotic lesions. The results have suggested that lncRNA NORAD attenuates endothelial cell senescence, endothelial cell apoptosis, and atherosclerosis via NF-κB and p53–p21 signaling pathways and IL-8, in which NORAD-mediated effect on IL-8 might through the direct interaction with SFPQ.

Flax seed meal (FSM) is rich in various nutrients, especially CP and energy, and can be used as animal protein feed. In animal husbandry production, it is a long-term goal to replace soybean meal (SBM) in animal feed with other plant protein feed. However, studies on the effects of replacing SBM with FSM in fattening sheep are limited. The aim of this experiment was to study the effects of replacing a portion of SBM with FSM on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep. Thirty-six Dorper × Small Thin-Tailed crossbred rams (BW = 40.4 ± 1.73 kg, mean ± SD) were randomly assigned into four groups. The dietary treatments (forage/concentrate, 45 : 55) were isocaloric according to the nutrient requirements of rams. Soybean meal was replaced with FSM at different levels (DM basis): (1) 18% SBM (18SBM), (2) 12% SBM and 6% FSM (6FSM), (3) 6% SBM and 12% FSM (12FSM) and (4) 18% FSM (18FSM). The rams were fed in individual pens for 60 days, with the first 10 days for adaptation to diets, and then the digestibility of nutrients was determined. There was no significant difference in DM intake, but quadratic (P < 0.001) effects on the average daily gain and feed efficiency were detected, with the highest values in the 6FSM and 12FSM groups. For DM and NDF digestibility, quadratic effects were observed with the higher values in the 6FSM and 12FSM groups, but the digestibility of CP linearly decreased with the increase in FSM in the diet (P = 0.043). There was a quadratic (P < 0.001) effect of FSM inclusion rate on the estimated microbial CP yield. However, the values of intestinally absorbable dietary protein decreased linearly (P < 0.001). For the supply of metabolisable protein, both the linear (P = 0.001) and quadratic (P = 0.044) effects were observed with the lowest value in the 18FSM group. Overall, the results indicated that SBM can be effectively replaced by FSM in the diets of fattening sheep and the optimal proportion was 12.0% under the conditions of this experiment.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.

Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes., Hepatic gluconeogenesis is important for glucose homeostasis and a therapeutic target for type 2 diabetes. Here, the authors show that the RNA-binding adaptor protein Sam68 promotes the expression level of gluconeogenic genes and increases blood glucose levels by stabilizing the transcriptional coactivator CRTC2, while hepatic Sam68 deletion alleviates hyperglycemia in mice.

Some grain processing by-products rich in digestible fiber are good feed resources for ruminants. This experiment was conducted to investigate the effects of replacing a portion of corn and corn stover with the combinations of corn bran and soybean hulls in the diet of fattening lambs on nutrient digestion, rumen microbial protein synthesis, and growth performance. A total of 36 Dorper × Small Thin-Tailed crossbred ram lambs (BW = 22.2 ± 0.92 kg; mean ± SD) were randomly divided into three groups, and each group was fed 1 of 3 treatment diets: 1) 0% corn bran and soybean hulls (control); 2) 9% corn bran and 9% soybean hulls (18MIX); and 3) 17% corn bran and 17% soybean hulls (34MIX). The feeding experiment was conducted for 70 days, with the first 10 days for adaption. The DM intake was higher for 34MIX (1635.3 g/d) than for control diet (1434.7 g/d; P = 0.001). Lambs fed 18MIX and 34MIX diets (230.2 and 263.6 g/d, respectively) had higher average daily gain and feed efficiency than those fed control diet (194.8 g/d; P 0.05). The metabolizable protein supplies were improved with increasing co-products inclusion rate. The results indicated that corn bran and soybean hulls in combination can effectively replace a portion of corn and corn stover in the ration of finishing lambs with positive effect on nutrient digestion and growth performance.

Aims The imbalance of M1/M2 macrophage ratio promotes the occurrence of diabetic cardiomyopathy (DCM), but the precise mechanisms are not fully understood. The aim of this study was to investigate whether miR-471-3p/silent information regulator 1 (SIRT1) pathway is involved in the macrophage polarization during the development of DCM. Methods Immunohistochemical staining was used to detect M1 and M2 macrophages infiltration in the heart tissue. Flow cytometry was used to detect the proportion of M1 and M2 macrophages. Expression of miR-471-3p was quantified by real time quantitative-PCR. Transfection of miRNA inhibitor into RAW264.7 cells was performed to investigate the underlying mechanisms. Bioinformatics methods and western blotting were used to explore the target gene of miR-471-3p and further confirmed by dual luciferase reporter assay. Key findings We observed that M1 macrophages infiltration in the heart of tissue in DCM while M2 type was decreased. M1/M2 ratio was increased significantly in bone marrow-derived macrophages (BMDMs) from db/db mice and in RAW264.7 cells treated with advanced glycation end products (AGEs). Meanwhile, miR-471-3p was significantly upregulated in RAW264.7 cells induced by AGEs and inhibition of miR-471-3p could reduce the inflammatory polarization of macrophages. Bioinformatics analysis identified SIRT1 as a target of miR-471-3p. Both dual luciferase reporter assay and western blotting verified that miR-471-3p negatively regulated SIRT1 expression. SIRT1 agonist resveratrol could downregulate the increased proportion of M1 macrophages induced by AGEs. Conclusion Our results indicated that the development of DCM was related to AGEs-induced macrophage polarized to M1 type through a mechanism involving the miR-471-3p/SIRT1 pathway.

Background Oxidative stress induces mitochondrial dysfunction, causing memory loss. Long noncoding RNAs influence mitochondrial function and suppress oxidative stress by regulating target protein expression and gene transcription. Celastrol, a natural antioxidant extracted from Tripterygium wilfordii Hook F. (“Thunder of God Vine”), effectively alleviates oxidative stress-mediated tissue injury. In the present study, we examined the effects of celastrol on memory dysfunction induced by ischemia/reperfusion (I/R) and elucidated the mechanisms underlying these effects. Methods C57BL/6 mice were used to mimic I/R using the bilateral common carotid clip reperfusion method, and a hippocampal cell line (HT-22) cells were used to establish a model of oxygen–glucose deprivation/reoxygenation (OGD/R). We observed changes in behavior and mitochondrial structure. Cell activity, cell respiration, and antioxidant capacity were measured. MAP3K12, p-JNK, p-c-Jun, p-Akt/Akt, PI3K, Bcl-2, and Bax expression were evaluated. Results I/R or OGD/R significantly increased AK005401 and MAP3K12 expression, further attenuating PI3K/Akt activation, promoting reactive oxygen species generation and causing mitochondrial dysfunction and cell apoptosis, thereby resulting in memory dysfunction. Celastrol increased antioxidant capacity, inhibited cell apoptosis, and improved mitochondrial function, effectively improving learning and memory by downregulating AK005401 and MAP3K12 and activating PI3K/Akt. Conclusions The AK005401/MAP3K12 signaling pathway has an important role in I/R-mediated hippocampal injury, and celastrol can potentially reduce or possibly prevent I/R-induced neuronal injury by downregulating AK005401/MAP3K12 signaling.

Background: Short-term melatonin treatment of cashmere goats has been shown to increase the quantity and quality of cashmere fibre. However, the long-term effects of melatonin treatment on cashmere production and hair follicle characteristics of these goats has not been reported. Therefore, we investigated changes in cashmere production and in hair follicle populations and their activity in melatonin-treated cashmere goats for two consecutive cashmere growth cycles.Methods: Twenty-four female Inner Mongolian Cashmere goats were randomly allocated to two groups (n = 12), one of which received melatonin implants, the other being an untreated control group. Melatonin implants were subcutaneously inserted behind the ear at a dose of 2 mg/kg live weight on two occasions – 30 April and 30 June 2016. Cashmere samples were collected at combing in April of 2017 and 2018. Blood and skin samples were collected monthly between April and September 2016, and during April and September in 2017. Results: Our results showed that melatonin treatment of cashmere goats in one cashmere growth cycle increased (P < 0.05) weight, length, density and decreased (P < 0.01) diameter of cashmere fibre, however it did not affect cashmere production in the following annual cycle. Melatonin treatment of cashmere goats had no effect on the population of secondary hair follicles for the two consecutive cycles. However, in the first growth cycle following treatment it advanced (P < 0.05) the onset of activity of secondary hair follicles by 2 months and it increased (P < 0.05) the population of these follicles that were active. Serum melatonin concentrations of the treated goats were elevated, relative to those of the control group (P < 0.05), but only during the first growth cycle.Conclusions: In summary, melatonin treatment of cashmere goats in one cashmere growth cycle improved cashmere production for that cycle only, with no residual effects on the following cycle. This makes the technique acceptable to the cashmere goat industry. The improvement in cashmere production following treatment of goats with melatonin appears to involve an acceleration of the annual regeneration of secondary hair follicles and an increased population of active secondary hair follicles in the skin of cashmere goats.

Neutral detergent fiber (NDF), as the main component of structural carbohydrate in forage, is a vital factor impacting the performance of young ruminants. The objective of this study was to evaluate the effect of starter NDF level on the rumen fermentation and morphometrics of lambs fed isocaloric and isonitrogenous diets. One hundred Hu sheep lambs (18 days of age and 6.1 ± 0.1 kg body weight [BW]) were randomly assigned to one of the four starter diets differing in their NDF levels on a dry matter (DM) basis: 1) diet with 14% NDF (NDF14), 2) diet with 18% NDF (NDF18), 3) diet with 22% NDF (NDF22), and 4) diet with 26% NDF (NDF26). Lambs were fed milk replacer with designed starters from 21 to 60 d of age and then transitioned to the same starter feed until 90 d of age. Six lambs from each treatment (close to the average BW of the corresponding treatment) were selected and slaughtered at 90 d of age for measuring rumen fermentation, anatomical development, and morphometric characteristics. Lambs receiving NDF22 and NDF26 starter diets had greater (P < 0.05) intake of starter and total DM, and average daily gain during the overall period, thereby heavier (P < 0.05) final weights at 90 d of age compared with those fed NDF14 starter diet. At 90 d of age, lambs fed NDF22 and NDF26 starters had higher rumen pH, followed by lower propionate, higher acetate concentrations (P < 0.05), and a higher acetate-to-propionate ratio compared with those fed NDF14 and NDF18 starters (P < 0.05). No significant differences of the full and empty weight of reticulorumen were observed among treatments; however, the reticulorumen weight expressed as a percentage of the complex stomach weight was lower (P < 0.05) in lambs fed NDF22 and NDF26 starters compared with those fed NDF14 starter. Furthermore, NDF14 lambs had the thickest keratin layer and epithelium compared with those in the other treatments but showed the thinnest muscle layer (P < 0.05). These results suggest that increasing starter NDF levels can ameliorate the rumen fermentation environment and alleviate hyperkeratosis and plaque formation in the rumen epithelium. In summary, including 22% and 26% NDF in the pelleted starter can improve the performance of lambs.

Increased aerobic glycolysis in endothelial cells of atheroprone areas of blood vessels has been hypothesized to drive increased inflammation and lesion burden but direct links remain to be established. Here we show that endothelial cells exposed to disturbed flow in vivo and in vitro exhibit increased levels of protein kinase AMP-activated (PRKA)/AMP-activated protein kinases (AMPKs). Selective deletion of endothelial Prkaa1, coding for protein kinase AMP-activated catalytic subunit alpha1, reduces glycolysis, compromises endothelial cell proliferation, and accelerates the formation of atherosclerotic lesions in hyperlipidemic mice. Rescue of the impaired glycolysis in Prkaa1-deficient endothelial cells through Slc2a1 overexpression enhances endothelial cell viability and integrity of the endothelial cell barrier, and reverses susceptibility to atherosclerosis. In human endothelial cells, PRKAA1 is upregulated by disturbed flow, and silencing PRKAA1 reduces glycolysis and endothelial viability. Collectively, these results suggest that increased glycolysis in the endothelium of atheroprone arteries is a protective mechanism., Increased glycolysis and inflammatory responses have been observed in endothelial cells exposed to disturbed flow. However, the role of endothelial glycolysis in atherosclerosis is unclear. Here the authors unveil a protective role for glycolysis by showing that endothelial deletion of Prkaa1 accelerates atherosclerosis in hyperlipidemic mice through a reduction of glycolytic metabolism.

Ketamine has a rapid, obvious, and persistent antidepressant effect, but its underlying molecular mechanisms remain unknown. Recently, microRNAs (miRNAs) have emerged as important modulators of ketamine’s antidepressant effect. We investigated the alteration in miR-29b-3p in the brain of rats subjected to ketamine administration and chronic unpredictable mild stress (CUMS), and a sucrose preference test and forced swimming test were used to evaluate the rats’ depressive-like state. We used recombination adeno-associated virus (rAAV) or lentivirus-expressing miR-29b-3p to observe the change in metabotropic glutamate receptor 4 (GRM4). Cell culture and electrophysiological recordings were used to evaluate the function of miR-29b-3p. Ketamine dramatically increased miR-29b-3p expression in the prefrontal cortex of the normal rats. The dual luciferase reporter test confirmed that GRM4 was the target of miR-29b-3p. The miR-29b-3p levels were downregulated, while the GRM4 levels were upregulated in the prefrontal cortex of the depressive-like rats. The ketamine treatment increased miR-29b-3p expression and decreased GRM4 expression in the prefrontal cortex of the depressive-like rats and primary neurons. By overexpressing and silencing miR-29b-3p, we further validated that miR-29b-3p could negatively regulate GRM4. The silencing of miR-29b-3p suppressed the Ca2+ influx in the prefrontal cortex neurons. The miR-29b-3p overexpression contributed to cell survival, cytodendrite growth, increases in extracellular glutamate concentration, and cell apoptosis inhibition. The overexpression of miR-29b-3p by rAAV resulted in a noticeable relief of the depressive behaviors of the CUMS rats and a lower expression of GRM4. The miR-29b-3p/GRM4 pathway acts as a critical mediator of ketamine’s antidepressant effect in depressive-like rats and could be considered a potential therapeutic target for treating major depression disorder., Ketamine: Insights into antidepressant effects Changes in the levels of a microRNA molecule that regulates the activity of a specific gene may be responsible for some of the antidepressant effects of the drug ketamine. Ketamine is used as a tranquilizer and anesthetic, especially in veterinary practice, but there is increasing interest in its antidepressant action in humans. Researchers in China and USA led by Xiao-Bin Wang at the Affiliated Hospital of Southwest Medical University in Luzhou, China, found that ketamine dramatically raised the levels of a specific microRNA in the prefrontal cortex region of rats’ brains. This effect was linked to the ability of the microRNA to inhibit a gene and thus decrease the production of a receptor protein in brain cell membranes. The molecular signaling pathway this research has identified could be a target for new antidepressant therapies.

To investigate the effects of maternal dietary selenium (Se-enriched yeast) on testis development, testosterone level and steroidogenesis-related gene expression in testis of their male kids, selected pregnant Taihang Black Goats were randomly allotted to four treatment groups. They were fed the basal gestation and lactation diets supplemented with 0 (control), 0.5, 2.0 and 4.0 mg of Se/kg DM. Thirty days after weaning, testes were collected from the kids. After the morphological development status of testis was examined, tissue samples were collected for analyzing testosterone concentration and histological parameters. Testosterone synthesis-related genes were detected using real-time PCR. Localization and quantification of androgen receptor (AR) in testis of goats were determined by immunohistochemical and western blot analysis. The results show that Se supplementation in the diet of dams led to higher (p < 0.05) testicular weight, volume, length, width, transverse and vertical grith of their male kids. Excessive Se (4.0 mg/kg) can inhibit the development of testis by decreasing testicular weight and volume. The density of spermatogenic cells and Leydig cells in the Se treatment groups was significantly (p < 0.05) higher than that in the control. Maternal dietary Se did not affect the thickness of testes, thickness of germinal epithelium and diameter of seminiferous tubule. Se supplemented in the diet of dams improved the testosterone level in testis tissue and serum, and promote the expression of testosterone-related genes. The mRNA expression of StAR, 3β-HSD and CYP11A1 was decreased with the increasing dietary Se levels of dams. Maternal dietary Se can improve the AR protein abundance in testis of their offspring. AR immunopositive product was detected in Leydig cells, peritubular myoid cells, perivascular smooth muscle cells, primary spermatocytes and spermatids. The expression of AR in spermatogenetic cells is stage specific. This study suggests that maternal dietary Se can influence the testis development and spermatogenesis of their male kids by modulating testosterone synthesis in goats. More attention should be given to the potential role of maternal nutrition in improving reproductive performance of their offspring.

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.

The objective of this study was to investigate the effect of dietary Tartary buckwheat extract (TBE) supplementation on animal growth performance, meat quality and antioxidative activity in the Longissimus thoracis et lumborum muscle of lambs. The results showed that dietary TBE increased body weight, average daily gain, carcass weight, dry matter intake, and digestive organ weight. Dietary TBE had no effect on the pH, color, shear force or intramuscular fat of Longissimus muscle examined, whereas the cooking loss was decreased. The total antioxidative capacity and glutathione peroxidase 4 (GPx4) activity of Longissimus muscle were increased in lambs fed TBE. The mRNA contents of superoxide dismutase, catalase, GPx4 and nuclear factor-like-2 factor (Nrf2) did not vary among the groups, and greater protein levels of GPx4 and Nrf2 were observed. Taken together, these results suggest that TBE can be used as a feed ingredient in lamb production to improve its growth performance, and relieve oxidative stress and increase water holding capacity of meat.

Previous studies reported that miR-146a was involved in small intestine ischemia-reperfusion (I/R) injury, but the mechanism is largely vague. Here, we aimed to identify the change of miR-146a in patients with mesenteric ischemia and explore the potential regulatory mechanism of miR-146a in intestine epithelial cells survival under ischemia and I/R injury. The plasma of 20 patients with mesenteric ischemia and 25 controls was collected to examine the miR-146a expression by qPCR. Rat intestinal epithelial cells (IEC-6) and 24 male Sprague-Dawley rats were included to build ischemia and I/R model in vitro and in vivo. The qPCR results showed that miR-146a decreased both in the plasma of patients with mesenteric ischemia and in IEC-6 cells and rat small intestine tissues in ischemia and I/R model compared to controls. Both the in vitro and in vivo results showed that I/R resulted in more severe apoptotic injury than ischemia. Cleaved-caspase 3, TLR4, TRAF6, and nuclear NF-κB p65 were up-regulated accompanying reduced XIAP and SOCS3 expression in intestinal ischemia and I/R injury. After up-regulation of miR-146a in IEC-6 cells, increased cell survival and decreased cell apoptosis were observed, concomitant with decreased cleaved-caspase 3 and down-regulated TLR4/TRAF6/NF-κB pathway. What is more, this protective effect was blocked by TRAF6 overexpression and increased nuclear NF-κB p65 nuclear. Taken together, this study revealed that miR-146a expression was decreased in small intestine ischemia and I/R injury. And miR-146a improves intestine epithelial cells survival under ischemia and I/R injury through inhibition TLR4, TRAF6, and p-IκBα, subsequently leading to decreased NF-κB p65 nuclear translocation.

Objective The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α–induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury. Approach and results The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68−/−) and WT mice. Sam68−/− mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68−/− mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α–induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68−/− vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction. Conclusions Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

// Zhou-Guang Wang 1, 2, 3, * , Hao Li 1, * , Yan Huang 2, * , Rui Li 2 , Xiao-Fan Wang 2 , Li-Xia Yu 2 , Xue-Qiang Guang 1 , Lei Li 1 , Hong-Yu Zhang 2 , Ying-Zheng Zhao 2 , Chunxiang Zhang 1 , Xiao-Kun Li 2, 3 , Rong-Zhou Wu 1 , Mao-Ping Chu 1 , Jian Xiao 1, 2 1 Institute of Cardiovascular Development and Translational Medicine, Children’s Heart Center, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325027, China 2 Molecular Pharmacology Research Center, School of Pharmacy, Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou 325035, China 3 Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, Changchun, 130012, China * These authors have contributed equally to this work Correspondence to: Jian Xiao, email: xfxj2000@126.com Mao-Ping Chu, email: chmping@126.com Rong-Zhou Wu, email: wrz71@hotmail.com Keywords: NGF, myocardial ischemia/reperfusion, autophagy, PI3K/Akt/mTOR, ubiquitin Received: April 14, 2016 Accepted: December 06, 2016 Published: December 27, 2016 ABSTRACT The dysregulation of autophagy is related to a variety of cardiovascular diseases, such as myocardial ischemia/reperfusion (I/R). Nerve growth factor (NGF) has been shown to have therapeutic potential in ischaemic heart injury. In this study, we demonstrate that NGF administration can accelerate autophagic flux and attenuate protein ubiquitination in myocardial I/R heart. Our results showed that NGF could restored heart function and decreased the apoptosis of cardiomyocytes which induced by myocardial I/R injury. The protective effect of NGF is associated with the inhibition of autophagy related proteins. On another hand, NGF enhances the clearance of ubiquitinated protein and increases the survival of myocardial cell in vivo and in vitro . Additionally, NGF could activate the PI3K/AKT and mTOR signaling pathways. These results suggested that the cardioprotective effect of NGF is related to the restoration of autophagic flux and attenuation of protein ubiquitination via the activation of PI3K/AKT and mTOR pathway.

Both HIV and antiretroviral therapy could induce vascular aging with unclear mechanisms. In this study, via microarray analysis, we identified, for the first time, that miR-34a expression was significantly increased in both HIV-infected, and antiretroviral agents-treated vessels and vascular endothelial cells (ECs) from these vessels. In cultured ECs, miR-34a expression was significantly increased by HIV-Tat protein and by the antiretroviral agents, lopinavir/ritonavir. Both HIV-Tat protein and antiretroviral agents could induce EC senescence, which was inhibited by miR-34a inhibition. In contrast, EC senescence was exacerbated by miR-34a overexpression. In addition, the vascular ECs isolated from miR-34a knockout mice were resistant to HIV and antiretroviral agents-mediated senescence. In vivo, miR-34a expression in mouse vascular walls and their ECs was increased by antiretroviral therapy and by HIV-1 Tat transgenic approach. miR-34a inhibition could effectively inhibit both HIV-Tat protein and antiretroviral therapy-induced vascular aging in mice. The increased miR-34a was induced via p53, whereas Sirt1 was a downstream target gene of miR-34a in both HIV-Tat protein and antiretroviral agents-treated ECs and vessels. The study has demonstrated that miR-34a is a common link in both HIV and antiretroviral therapy-mediated vascular aging.

Sepsis poses a serious global health problem with an overall mortality rate of 30%, in which the vascular injury is a major contributor. The study is to determine the expression profile of micro-RNAs in endotoxic vascular walls and their potential roles in sepsis-related vascular injury.Prospective randomized study.Laboratory investigation.Male C57BL/6 mice, average weight 26.5 ± 1.8 g.Endotoxemia was induced in mice via lipopolysaccharide injection (20 mg/kg, intraperitoneal) (Sigma, St. Louis, MO). The control mice were injected with the same amount of saline (500 μL, intraperitoneal). In a subgroup of mice, a high dose of lipopolysaccharide (30 mg/kg, intraperitoneal) was applied to induce endotoxin-related death.The mi-RNA expression profiles in aortas from lipopolysaccharide-induced endotoxic mice were determined. The result demonstrated that some micro-RNAs were aberrantly expressed in endotoxic mouse arteries. Among them, the endothelial cell-enriched/endothelial cell-specific miR-126a-3p was significantly down-regulated in endotoxic mouse arteries, septic human vessels, as well as vascular endothelial cells isolated from endotoxic mice or treated with lipopolysaccharide. The down-regulation of miR-126a-3p occurred at transcriptional level via the decreased expression of Krüppel-like factor 2, which could be inhibited by Krüppel-like factor 2 over-expression via adenovirus expressing Krüppel-like factor 2. The down-regulation of miR-126a-3p in endothelial cells resulted in the increased apoptosis, and decreased proliferation and migration, which were inhibited by miR-126a-3p mimics. In vivo, over-expression of miR-126a-3p via lentivirus attenuated endotoxemia-induced injuries on endothelial function and vascular permeability. We found that SPRED1 and VCAM-1 were two direct target genes of miR-126a-3p related to miR-126a-3p-mediated effects in endotoxemia. Finally, the survival rate of endotoxic mice was significantly increased by the over-expression of miR-126a-3p.The results suggest that vascular micro-RNAs such as miR-126a-3p may represent novel mechanisms and new therapeutic targets for endotoxemia-induced vascular injury and endotoxic mortality.

Background Recent studies have shown that circular RNA (circRNA) is rich in microRNA (miRNA) binding sites. We have previously demonstrated that the antidepressant effect of ketamine is related to the abnormal expression of various miRNAs in the brain. This study determined the expression profile of circRNAs in the hippocampus of rats treated with ketamine. Methods The aberrantly expressed circRNAs in rat hippocampus after ketamine injection were analyzed by microarray chip, and we further validated these circRNAs by quantitative reverse-transcription PCR (qRT-PCR). The target genes of the different circRNAs were predicted using bioinformatic analyses, and the functions and signal pathways of these target genes were investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results Microarray analysis showed that five circRNAs were aberrantly expressed in rat hippocampus after ketamine injection (fold change > 2.0, p p = 0.03), while one was significantly reduced (rno_circRNA_005442; fold change = 0.37; p = 0.01). We discovered a significant enrichment in several GO terms and pathways associated with depression. Conclusion Our findings showed the abnormal expression of ketamine-induced hippocampal circRNAs in rats.

Brain stroke is one of the leading causes of death worldwide. We explored a potential stroke-related role for a newly found microRNA, miR-1247-3p, and one of its target genes, caspase-2, predicted by TargetScanVert. In the present study, we found that miR-1247-3p was downregulated during ischemia/reperfusion (I/R) and that LV-miR-1247-3p overexpression attenuated brain impairment induced by I/R. Similar results were observed in neuro2a (N2a) cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R). Caspase-2 was upregulated in the I/R and OGD/R model, while Z-VDVAD-FMK - the inhibitor of caspase-2-inhibited apoptosis of N2a cells induced by OGD/R. An miR-1247-3p mimic inhibited caspase-2 expression and attenuated apoptosis of N2a cells induced by OGD/R. Myocardin-related transcription factor-A (MRTF-A) overexpression upregulated miR-1247 and mature miR-1247-3p levels and attenuated apoptosis induced by OGD/R, whereas its anti-apoptotic function could be blocked by a miR-1247-3p inhibitor. Hence, we conclude that miR-1247-3p may protect cells during brain stroke. This study offers insights for the development of effective therapeutics for promoting the survival of cerebral neurons during brain I/R injury.

The study is to establish a novel method to determine the endothelial function in mouse carotid arteries in vivo by using high-resolution ultrasound images. Atherosclerosis in carotid arteries is induced in ApoE−/− mice with a Western diet. The ultrasound of the ventral neck generates clear pictures of the common carotid arteries. Acetylcholine at the range from 5 to 20 μg/kg/min (iv) is able to induce a dose-dependent relaxation as shown by the increased diameter of these normal mouse carotid arteries, which is impaired in atherosclerotic arteries. The endothelial function determined by ultrasound images in vivo matches well with that determined in isolated carotid arterial rings in vitro. All animals are survival after the endothelial function measurement. In this study, we have established a standard method to determine the mouse endothelial function in vivo. It is a reliable, safe and survival method that could be used repetitively in mouse arteries.

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes.

Background/Aims: Acute myocardial infarction is a serious disease with high morbidity and mortality. microRNAs (miRNAs) have been proved to play an important role in modulating myocardial ischemia and reperfusion injury. Hence, in this study, we constructed H9c2 cell model to elucidate the roles of microRNA-486 (miR-486) in preventing hypoxia-induced damage in H9c2 cells. Methods: H9c2 cells were cultured in hypoxic incubator with 1% O2 to simulate hypoxia and/or transfected with miR-486 mimic, scramble, anti-miR-486, si-N-myc downstream-regulated gene 2 (NDRG2) and their corresponding negative controls (NC). Effects of miR-486 and/or NDRG2 dysregulation on hypoxia-induced myocardial injury in H9c2 cells were investigated by evaluating cell viability, migration, invasion and apoptosis using Cell Counting Kit-8 (CCK-8), transwell assay, flow cytometry, respectively. The proteins expression and RNA expression were detected by western blot and quantitative real time polymerase chain reaction (qRT-PCR), respectively. Results: Hypoxia treatment induced damage in H9c2 cells by decreasing cell viability, migration and invasion and increasing cell apoptosis. Moreover, hypoxia inhibited the expression of miR-486 in H9c2 cells. Overexpression of miR-486 alleviated hypoxia-induced myocardial injury in H9c2 cells, while suppression of miR-486 further aggravated hypoxia-induced injury. Furthermore, NDRG2 expression was negatively regulated by miR-486, and NDRG2 was confirmed as a target of miR-486. Knockdown of NDRG2 alleviated the effects of miR-486 suppression on hypoxia-induced myocardial injury. Besides, knockdown of NDRG2 markedly inhibited the activation of c-Jun N-terminal kinase (JNK) /c-jun and nuclear factor κB (NF-κB) signaling pathways in hypoxia-induced H9c2 cells. Conclusion: Our findings indicate that miR-486 may alleviate hypoxia-induced myocardial injury possibly by targeting NDRG2 to inactivate JNK/c-jun and NF-κB signaling pathways. miR-486 may be a potential target for treating ischemic myocardial injury following acute myocardial infarction.

Normal high density lipoprotein (HDL) protects vascular function; however these protective effects of HDL may absent in valvular heart disease (VHD). Because vascular function plays an important role in maintaining the circulation post-cardiac surgery and some patients are difficult to stabilize, we hypothesized that a deleterious vascular effect of HDL may contribute to vascular dysfunction in VHD patients following surgery. HDL was isolated from age-match 28 healthy subjects and 84 patients with VHD and during cardiac surgery. HDL pro-inflammation index was measured and the effects of HDL on vasodilation, protein interaction, generation of nitric oxide (NO) and superoxide were determined. Patients with VHD received either simvastatin (20mg/d) or routine medications, and endothelial effects of HDL were characterized. HDL inflammation index significantly increased in VHD patients and post-cardiac surgery. HDL from VHD patients and post-cardiac surgery significantly impaired endothelium-dependent vasodilation, inhibited both Akt and endothelial nitric oxide synthase (eNOS) phosphorylation at S1177, eNOS associated with heat shock protein 90 (HSP90), NO production and increased eNOS phosphorylation at T495 and superoxide generation. Simvastatin therapy partially reduced HDL inflammation index, improved the capacity of HDL to stimulate eNOS and Akt phosphorylation at S1177, eNOS associated with HSP90, NO production, reduced eNOS phosphorylation at T495 and superoxide generation, and improved endothelium-dependent vasodilation. Our data demonstrated that HDL from VHD patients and cardiac surgery contributed to endothelial dysfunction through uncoupling of eNOS. This deleterious effect can be reversed by simvastatin, which improves the vasoprotective effects of HDL. Targeting HDL may be a therapeutic strategy for maintaining vascular function and improving the outcomes post-cardiac surgery.

Ischemia exerts a negative impact on mitochondrial function, which ultimately results in neuronal damage via alterations in gene transcription and protein expression. Long non- coding RNAs (LncRNAs) play pivotal roles in the regulation of target protein expression and gene transcription. In the present study, we observed the effect of an unclassical LncRNA AK005401on ischemia/reperfusion (I/R) ischemia-mediated hippocampal injury and investigated the regulatory role of fibroblast growth factor 21 (FGF21) and Yin Yang 1 (YY1). C57Black/6 mice were subjected to I/R using the bilateral common carotid clip reperfusion method, and AK005401 siRNA oligos were administered via intracerebroventricular injection. HT22 cells were used to establish a model of oxygen-glucose deprivation/reoxygenation (OGD/R). We observed pathological morphology and mitochondrial structure. Neuronal apoptosis was evident. Cell activity, cell respiration, FGF21, YY1, and antioxidant capacity were evaluated. I/R or OGD/R significantly increased the expressions of AK005401and YY1 and decreased FGF21expression, which further attenuated the activation of PI3K/Akt, promoted reactive oxygen species (ROS) generation, and then caused mitochondria dysfunction and cell apoptosis, which were reversed by AK005401 siRNA oligos and were aggravated by overexpression of AK005401 and YY1. We conclude that AK005401/YY1/FGF21 signaling pathway has an important role in I/R-mediated hippocampal injury.

Supercooling sperm in liquid nitrogen vapour is a feasible and economic technique for the practical production. The study aimed to reveal the negative effects of this rapid freezing and thawing processes on Taihang black goat spermatozoa and to find out the changing of spermatozoa motility and ultrastructure by using CASA and TEM. Qualified semen samples, which collected from twenty Chinese Taihang black goats using artificial vagina were pooled and investigated the kinematics parameters and ultrastructural morphology. The results showed that freezing-thawing caused a significant reduction in the spermatozoon total motility (P0.001), in rapid and medium cell numbers (P0.001) and motility parameters (VAP, VSL, VCL, ALH and BCF) (P0.01). Immotile spermatozoa number was increased significantly after freezing-thawing (P0.001). In the ultrastructural analysis, the shape with a sperm nucleus characterized by ruptures, bend and deformity was observed. The plasma membranes were broken, and nucleoplasm erupted. The mitochondria in the middle piece were disturbed by partial absence or additional accumulations. Swelling, coiling, vacuolization and structural disorganization of mitochondria were also observed. In conclusion, Freezing-thawing procedure has a detrimental effect on motility, membrane integrity and mitochondria of goat spermatozoa. Transmission electron microscopy provides an intuitive observation to investigate deformity spermatozoa.

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR-382 was down-regulated in alcohol-treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo, we identified that the dopamine receptor D1 (Drd1) is a direct target gene of miR-382. Via this target gene, miR-382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR-382 significantly attenuated alcohol-induced up-regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1-induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism.

Background Kawasaki disease ( KD ) is now the most common cause of acquired cardiac disease in children due to permanent coronary artery damage with unknown etiology. The study sought to determine the role of blood micro RNA miR‐223 in KD and KD ‐induced injuries in vascular endothelial cells ( EC s) as well as the mechanisms involved. Methods and Results Micro RNA profiles in serum from patients with KD and from healthy controls were assessed by microarray analysis. We noted that multiple serum micro RNA s were aberrantly expressed in KD , among them miR‐223, which was the most upregulated abundant serum micro RNA . We found that bone marrow–derived blood cells (leukocytes and platelets) were able to secrete miR‐223 into serum. Vascular EC s had no endogenous miR‐223; however, the blood cell–secreted serum miR‐223 could enter into the vascular EC s in the vascular walls. The exogenous miR‐223 had strong biological effects on EC functions via its target genes such as IGF 1R. Interestingly, KD ‐induced EC injuries were related to increased miR‐223 because they were inhibited by miR‐223 knockdown. Finally, these observations were verified using miR‐223 knockout mice and the chimeric mice generated by transplantation of bone marrow from miR‐223 knockout mice into wild‐type mice. Conclusions In KD patients, the levels of blood cell–derived miR‐223 in EC s are significantly increased. The increased miR‐223 in EC s could work as a novel endocrine genetic signal and participate in vascular injury of KD . MiR‐223 may provide a novel mechanism and a new therapeutic target for vascular complication of KD .

Objective— On the luminal surface of injured arteries, platelet activation and leukocyte–platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor-κB is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-κB pathway in forming arterial neointima after arterial injury. Methods and Results— We performed carotid artery wire injuries in low-density lipoprotein receptor-deficient (LDLR –/– ) mice with a platelet-specific deletion of IκB kinase-β (IKKβ) (IKKβ fl/fl /PF4 cre /LDLR –/– ) and in control mice (IKKβ fl/fl /LDLR –/– ). The size of the arterial neointima was 61% larger in the IKKβ fl/fl /PF4 cre /LDLR –/– mice compared with the littermate control IKKβ fl/fl /LDLR –/– mice. Compared with the control mice, the IKKβ fl/fl /PF4 cre /LDLR –/– mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ibα shedding after platelet activation was compromised in the IKKβ-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17, the key enzyme involved in mediating glycoprotein Ibα shedding in activated IKKβ-deficient platelets. Conclusion— Platelet IKKβ deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-κB–related inhibitors should be carefully evaluated for use in patients after an arterial intervention.

Wine grape pomace (WGP) contains a rich source of polyphenols that can act as powerful antioxidants. The objective of this study was to investigate the effect of dietary WGP supplementation on antioxidative activity and epididymal sperm quality in rams. The rams were raised either under free-range or pen conditions, and the pen-raised rams were fed a WGP-containing diet (0, 5% and 10% of dry matter basis) for 74 days. An increase in the concentrations of reactive oxygen species (ROS, P < 0.05) and malondialdehyde (MDA, P < 0.05) were observed in the testes of rams subjected to restraint stress, and dietary WGP supplementation effectively decreased their contents (P < 0.05). Restraint stress reduced both weight and volume of testes, and impaired sperm quality. Dietary WGP supplementation increased testes weight, sperm concentration, motility and acrosomal integrity, and decreased sperm deformity in pen-raised animals (P < 0.05). The total antioxidative capacity (T-AOC) and catalase activity were decreased in the testes of pen-raised lambs (P < 0.05), and T-AOC, catalase, glutathione peroxidase 4 (GPx4) and superoxide dismutase (SOD) activity were increased when rams were fed the WGP-containing diet (P < 0.05). With the exception of SOD and GPx4, the mRNA contents of catalase and nuclear factor-like-2 factor (Nrf2) did not vary among the groups, and greater protein levels of SOD, catalase and GPx4 were observed in WGP-treated lambs (P < 0.05). Taken together, these results suggest that WGP can be used as a feed ingredient in rams to alleviate restraint induced oxidative stress and improve epididymal sperm quality.

Previous research has demonstrated that the behaviors observed in chronic unpredictable mild stressed (CUMS) rats are similar to the symptoms of depressed patients and that the abnormal expression of cerebral microRNAs is associated with depressive disorder. However, little is known regarding the expression profile of microRNAs induced by CUMS. In this study, we aimed to examine the hippocampal microRNA expression profile in CUMS rats. Forty adolescent male Sprague-Dawley rats were randomly divided into normal and model groups. The rats in the model group were stimulated daily with randomly applied mild stressors from among 14 different mild stressors. The stressors were changed every day and were applied for 35 consecutive days. On the 28th and 35th days after treatment, the weights, physical condition, sucrose preference, and open-field test scores of the rats of the two groups were evaluated. Successful induction of CUMS was considered if the differences of the above metrics between the two groups were statistically significant on the 28th and 35th days after treatment. Cerebral sucrose metabolism images of rats were obtained by 18F-FDG PET/CT. The rats were euthanized under anesthesia, and hippocampal tissues were collected for hematoxylin-eosin (HE) staining. In addition, the samples were used for microRNA array chip and qRT-PCR analysis. The target genes of different microRNAs were predicted using bioinformatic analysis, and the functions and signal pathways of these target genes were investigated by GO and KEGG analyses. Sixteen rats exhibited successful induction of CUMS. Cerebral 18F-FDG PET/CT imaging showed that the glucose metabolism rate of CUMS rats were significantly lower than normal rats in the central nucleus of the inferior colliculus (CIC, p = 0.022), the retrosplenial agranular area (RSA, p = 0.002), the second sensory cortex (S2, p = 0.028), the first auditory cortex (Au1, p = 0.012), the primary somatosensory cortex, barrel field (SIBF, p = 0.001), and the ventral posteromedial nucleus (VPM) of the right thalamus (p = 0.048). HE staining showed that hippocampal pyramidal cells CUMS rats were thinner, disordered, and exhibited irregular shapes, with many pyknotic cells. The microarray chip and qRT-PCR analysis revealed that five microRNAs were significantly up-regulated [miR-382-3p (p = 0.026), miR-183-5p (p = 0.018), miR-3573-5p (p = 0.042), miR-202-3p (p = 0.016), miR-493-3p (p = 0.009)], and only miR-370-3p was significantly down-regulated (p = 0.036). miRNA target gene prediction and functional annotation analysis showed significant enrichment in several GO terms and pathways associated with depression. Our findings provide supportive evidence for the abnormal expression of multiple CUMS-induced hippocampal microRNAs in rats as well as the involvement of these microRNAs in depressive disorder.

Many cardiac aging studies are performed on mice first and then, due to difficulty in mouse cardiomyocyte culture, applied the rat neonatal cardiomyocytes to further determine the mechanisms in vitro. Now, the technological challenge of mouse cardiomyocyte culture has been overcome and there is an increasing need for the senescence models of mouse cardiomyocytes. In this study, we have demonstrated that the senescence of mouse cardiomyocytes occurred with the extended culture time as shown by the increased β-galactosidase staining, increased p53 expression, decreased telomere activity, shorted telomere length, increased production of ROS, increased cell apoptosis, and impaired mitochondrial ΔΨm. These senescent responses shared similar results in aged mouse heart tissues in vivo. In summary, we have established and characterized a novel senescence model of mouse cardiomyocytes induced by the extended culture time in vitro. The cell model could be useful for the increased cardiac aging studies worldwide.

In this experiment the effect of maternal dietary selenium on the expression of Sel P and apoER2 of goat offspring was studied. The experiment was conducted on 119 Taihang Black Goats randomly divided into 4 groups which were fed with a basal diet, supplemented with 0 (control), 0.5, 2 and 4 mg kg −1 DM Se. Testis samples were collected from young male of each treatment group at the end of the study (30 d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. A significant decrease was observed in mRNA expression of Sel P and apoER2 in the testis of the Se-deficient (Group 1) and the Se-excess (Group 4) compared with that in Groups 2 and 3. A similar trend of the protein expression of Sel P and apoER2 was also found. These data indicate that maternal and dietary selenium has an effect on the expression of Sel P and apoER2 in testis of their offspring. In addition, both groups were similar suggesting that the relationship between Sel P and apoER2, and apoER2 is a receptor of Sel P in the seminiferous epithelium to uptake the selenium.

AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1(-/-)) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1(-/-) mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1(-/-) mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1(-/-) mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1(-/-) mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.

MicroRNAs (miRNAs) are a novel class of endogenous, small, noncoding RNAs that regulate gene expression via degradation, translational inhibition, or translational activation of their target messenger RNAs. Functionally, an individual miRNA is important as a transcription factor because it is able to regulate the expression of its multiple target genes. As a group, miRNAs are able to directly regulate at least 30% of genes in a cell. In addition, other genes may also be regulated indirectly by miRNAs. It is therefore not surprising that miRNAs could be the pivotal regulators in normal development, physiology, and pathology. Recent studies have identified that miRNAs are highly expressed in vasculature and their expression is dysregulated in diseased vessels. miRNAs are found to be critical modulators for vascular cell functions such as cell differentiation, contraction, migration, proliferation, and apoptosis. Accordingly, miRNAs are involved in the vascular dysfunction, ischemic angiogenesis, reendothelialization, and vascular neointimal lesion formation under diverse vascular diseases. miRNAs may serve as novel therapeutic targets for vascular diseases such as impaired angiogenesis or reendothelialization, restenosis, atherosclerosis, hypertension, and diabetic vascular complication. This review article summarizes the research progress regarding the roles of miRNAs in vascular diseases.

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

BACKGROUND: Protective mechanical ventilation with low tidal volume (V T ) and low plateau pressure reduces mortality and decreases the length of mechanical ventilation in patients with acute respiratory distress syndrome. Mechanical ventilation that will protect normal lungs during major surgical procedures of long duration may improve postoperative outcomes. We performed an animal study comparing 3 ventilation strategies used in the operating room in normal lungs. We compared the effects on pulmonary mechanics, inflammatory mediators, and lung tissue injury. METHODS: Female pigs were randomized into 3 groups. Group H-VT/3 (n = 6) was ventilated with a VT of 15 mL/kg predicted body weight (PBW)/positive end-expiratory pressure (PEEP) of 3 cm H 2 O, group L-VT/3 (n = 6) with a VT of 6 mL/kg PBW/PEEP of 3 cm H 2 O, and group L-VT/10 (n = 6) with a VT of 6 mL/kg PBW/PEEP of 10 cm H 2 O, for 8 hours. Hemodynamics, airway mechanics, arterial blood gases, and inflammatory markers were monitored. Bronchoalveolar lavage (BAL) was analyzed for inflammatory markers and protein concentration. The right lower lobe was assayed for mRNA of specific cytokines. The right lower lobe and right upper lobe were evaluated histologically. RESULTS: In contrast to groups H-VT/3 and L-VT/3, group L-VT/10 exhibited a 6-fold increase in inflammatory mediators in BAL (P < 0.001). Cytokines in BAL were similar in groups H-VT/3 and L-VT/3. Group H-VT/3 had a significantly lower lung injury score than groups L-VT/3 and L-VT/10. CONCLUSION: Comparing intraoperative strategies, ventilation with high PEEP resulted in increased production of inflammatory markers. Low PEEP resulted in lower levels of inflammatory markers. High VT/low PEEP resulted in less histologic lung injury.

Objective— Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. Methods and Results— Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A 2A receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A 2A receptor or α1–AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-κB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. Conclusion— Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

MicroRNA-21 (miR-21) is a highly expressed microRNA (miRNA) in cardiovascular system. Recent studies have revealed that its expression is deregulated in heart and vasculature under cardiovascular disease conditions such as proliferative vascular disease, cardiac hypertrophy and heart failure, and ischemic heart disease. miR-21 is found to play important roles in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. Accordingly, miR-21 is proven to be involved in the pathogenesis of the above-mentioned cardiovascular diseases as demonstrated by both loss-of-function and gain-of-function approaches. Programmed cell death 4 (PDCD4), phosphatase and tensin homology deleted from chromosome 10 (PTEN), sprouty1 (SPRY1), and sprouty2 (SPRY2) are the current identified target genes of miR-21 that are involved in miR-21-mediated cardiovascular effects. miR-21 might be a novel therapeutic target in cardiovascular diseases. This review article summarizes the research progress regarding the roles of miR-21 in cardiovascular disease.

Objective—To use the mice deficient in both adenosine receptor A2A(A2AR−/−) and apolipoprotein E (apoE−/−) to investigate the role of A2AR in mediating the interactions of leukocytes with injured arterial walls and the formation of arterial neointima induced by a guide wire.Methods and Results—In apoE−/−mice, A2AR deficiency increased the size of the arterial neointima in injured carotid arteries by 83%. Arterial neointima formation was also enhanced in chimeric mice that underwent bone marrow transplantation (these mice lacked A2AR in their bone marrow–derived cells). Epifluorescence intravital microscopy showed that neutrophil rolling and adherence to the injured arterial area were enhanced by 80% and 110% in A2AR−/−/apoE−/−mice, respectively. This phenomenon occurred even though the protein levels of homing molecules on A2AR-deficient neutrophils were unchanged from those of wild-type neutrophils. A2AR-deficient neutrophils exhibited an increase in the phosphorylation of p38 mitogen-activated protein kinase, P-selectin glycoprotein ligand-1 (PSGL-1) clustering, and the affinity of b2integrins. The inhibition of p38 phosphorylation abrogated the increased PSGL-1 clustering and β2integrin affinity, thus reversing the increased homing ability of A2AR-deficient leukocytes.Conclusion—A2AR plays a complex role in inflammation and tissue injury. The deficiency of A2AR enhances the homing ability of leukocytes and increases the formation of the arterial neointima after injury. A2AR antagonists are being tested for the treatment of neurodegenerative and other chronic diseases. An evaluation of the effect of A2AR antagonists on arterial restenosis after arterial angioplasty should be conducted.

MicroRNAs (miRNAs) have emerged as a novel class of endogenous, small, non-coding RNAs that negatively regulate over 30% of genes in a cell via degradation or translational inhibition of their target mRNAs. Functionally, an individual miRNA is important as a transcription factor because it is able to regulate the expression of its multiple target genes. Recent studies have identified that miRNAs are highly expressed in vasculature and their expression is deregulated in diseased vessels. miRNAs are found to be critical modulators for vascular cell functions such as cell differentiation, migration, proliferation, and apoptosis. Accordingly, miRNAs are involved in the angiogenesis and in the pathogenesis of vascular diseases. miRNAs may serve as novel biomarkers and therapeutic targets for vascular disease. This review article summarizes the research progress regarding the roles of miRNAs in vascular biology and vascular disease.

Acrylamide (ACR) and high contents of fat could be found co-existent in many foods processed by high temperature, such as deep-frying and roasting. This study investigated the effect of enhanced fat consumption on deficits of spermatogenesis induced by ACR, and explored potential mechanisms of oxidative damage involved in this pathology in mice. Results show that enhanced feeding of corn oil and pork fat on mice potentiated the decreases of spermatogonia along with mature sperms after treatment of ACR, and that spermatozoa quality is significantly reduced as a result of enhanced feeding of corn oil and pork fat on mice treated with ACR. Moreover, enhanced consumption of corn oil and pork fat potentiated the up-regulation of malondialdehyde (MDA) level in epididymal sperm and cauda epididymides, also up-regulated level of Protein carbonyls (PCOs) in cauda epididymides, of mice after treatment of ACR. Last, enhanced consumption of corn oil and pork fat potentiated the reduced activity of superoxide dismutases (SOD) in epididymal sperm, corpus, and cauda epididymides, also reduced activity of glutathione peroxidase (GPx) in cauda epididymides, of mice treated with ACR. These data suggest that enhanced feeding of corn oil and pork fat on mice potentiates ACR-induced oxidative stress in the epididymis and epididymal sperm and a subsequent effect on spermatogenesis.

Several recent reports have suggested that microRNAs (miRNAs) might play critical roles in acute myocardial infarction (AMI). However, the miRNA expression signature in the early phase of AMI has not been identified. In this study, the miRNA expression signature was investigated in rat hearts 6 h after AMI. Compared with the expression signature in the noninfarcted areas, 38 miRNAs were differentially expressed in infarcted areas and 33 miRNAs were aberrantly expressed in the border areas. Remarkably, miR-21 expression was significantly down-regulated in infarcted areas, but was up-regulated in border areas. The down-regulation of miR-21 in the infarcted areas was inhibited by ischemic preconditioning, a known cardiac protective method. Overexpression of miR-21 via adenovirus expressing miR-21 (Ad-miR-21) decreased myocardial infarct size by 29% at 24 h and decreased the dimension of left ventricles at 2 weeks after AMI. Using both gain-of-function and loss-of-function approaches in cultured cardiac myocytes, we identified that miR-21 had a protective effect on ischemia-induced cell apoptosis that was associated with its target gene programmed cell death 4 and activator protein 1 pathway. The protective effect of miR-21 against ischemia-induced cardiac myocyte damage was further confirmed in vivo by decreased cell apoptosis in the border and infarcted areas of the infarcted rat hearts after treatment with Ad-miR-21. The results suggest that miRNAs such as miR-21 may play critical roles in the early phase of AMI.

MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression. Functionally, an individual miRNA is as important as a transcription factor because it is able to regulate the expression of its multiple target genes. Recently, miR-221 and miR-222 have been found to play a critical role in cancer cell proliferation. However, their roles in vascular smooth muscle cell (VSMC) biology are currently unknown. In the present study, the time course changes and cellular distribution of miR-221 and miR-222 expression were identified in rat carotid arteries after angioplasty, in which their expression was upregulated and localized in VSMCs in the injured vascular walls. In cultured VSMCs, miR-221 and miR-222 expression was increased by growth stimulators. Knockdown of miR-221 and miR-222 resulted in decreased VSMC proliferation in vitro. Using both gain-of-function and loss-of-function approaches, we found that p27(Kip1) and p57(Kip2) were 2 target genes that were involved in miR-221– and miR-222–mediated effect on VSMC growth. Finally, knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and neointimal lesion formation after angioplasty. The results indicate that miR-221 and miR-222 are novel regulators for VSMC proliferation and neointimal hyperplasia. These findings may also represent promising therapeutic targets in proliferative vascular diseases.