• MMVD
• biomarkers
• canine
• chronic valvular disease
• miRNA
• predictive modeling

• Spanish Ministry of Science and Innovation

• cancer
• canine
• developmental disease
• infectious diseases
• inflammatory disease
• miRNA
• microRNA
• neoplasia

The complement system plays a vital role in myocardial ischemia/reperfusion (I/R) injury. microRNA (miR)-499 is involved in the cardioprotection of ischemic postconditioning (IPostC). The present study aimed to study the role of the complement system and miR-499 in IPostC. Rat hearts were subjected to coronary ligation for 30 min, followed by reperfusion for 2 h. IPostC was introduced at the onset of reperfusion with three cycles of reperfusion for 30 sec and coronary artery occlusion for 30 sec. To study the role of miR-499 in IPostC, adeno-associated virus (AAV) vectors of miR-499-5p (AAV-miR-499-5p) and miR-499-5p-sponge (AAV-miR-499-5p-sponge) were transfected via tail vein injection, followed by IPostC protocols. Cardiac injury as well as the status of local and systemic complement activation and inflammation were assessed. IPostC significantly attenuated I/R-induced rat cardiomyocyte apoptosis and the myocardial infarct size. These beneficial effects were accompanied by decreased local and circulating complement component (C)3a and C5a levels, decreased inflammatory marker expression, decreased NF-κB signaling and increased cardiac miR-499 expression. AAV-miR-499-5p prevented local and systemic complement activation and inflammation as well as enhanced the cardioprotection of IPostC, whereas AAV-miR-499-5p-sponge produced the opposite effects. In summary, IPostC protected the rat myocardium against I/R injury, by inhibiting local and systemic complement activation; inflammation; NF-κB signaling; and upregulation of miR-499. As such, miR-499 may have a critical role in IPostC-mediated cardioprotection against I/R injury.

• inflammation
• miR-499
• myocardial ischemia/reperfusion injury
• postconditioning
• the complement system

Toll-like receptor 2 (TLR2)-mediated myocardial inflammation serves an important role in promoting myocardial ischemic/reperfusion (I/R) injury. Previous studies have shown that miR-499 is critical for cardioprotection after ischemic postconditioning (IPostC). Therefore, the present study evaluated the protective effect of IPostC on the myocardium by inhibiting TLR2, and also assessed the involvement of microRNA (miR)-499. Rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The IPostC was 3 cycles of 30 sec of reperfusion and 30 sec of re-occlusion prior to reperfusion. In total, 90 rats were randomly divided into six groups (n=15 per group): Sham; I/R; IPostC; miR-499 negative control adeno-associated virus (AAV) vectors + IPostC; miR-499 inhibitor AAV vectors + IPostC; and miR-499 mimic AAV vectors + IPostC. It was identified that IPostC significantly decreased the I/R-induced cardiomyocyte apoptotic index (29.4±2.03% in IPostC vs. 42.64±2.27% in I/R; P<0.05) and myocardial infarct size (48.53±2.49% in IPostC vs. 66.52±3.1% in I/R; P<0.05). Moreover, these beneficial effects were accompanied by increased miR-499 expression levels (as demonstrated by reverse transcription-quantitative PCR) in the myocardial tissue and decreased TLR2, protein kinase C (PKC), interleukin (IL)-1β and IL-6 expression levels (as demonstrated by western blotting and ELISA) in the myocardium and serum. The results indicated that IPostC + miR-499 mimics significantly inhibited inflammation and the PKC signaling pathway and enhanced the anti-inflammatory and anti-apoptotic effects of IPostC. However, IPostC + miR-499 inhibitors had the opposite effect. Therefore, it was speculated that IPostC may have a miR-499-dependent cardioprotective effect. The present results suggested that miR-499 may be involved in IPostC-mediated ischemic cardioprotection, which may occur via local and systemic TLR2 inhibition, subsequent inhibition of the PKC signaling pathway and a decrease in inflammatory cytokine release, including IL-1β and IL-6. Moreover, these effects will ultimately lead to a decrease in the myocardial apoptotic index and myocardial infarct size via the induction of the anti-apoptotic protein Bcl-2, and inhibition of the pro-apoptotic protein Bax in myocardium.

• cardioprotection
• cardiopulmonary bypass
• ischemia/reperfusion
• microRNA

• Cardioprotection
• Dexmedetomidine
• Hypoxia/reoxygenation injury
• Med13
• Wnt/β-catenin signaling pathway
• miR-208b-3p

• Apoptosis
• Myocardial ischemia reperfusion
• Panax notoginseng saponins
• miR-30c-5p
• microRNAs

• CD38
• H9c2 cardiomyocytes
• apoptosis
• miR-499a-5p

• NLRP3
• adrenal injury
• cardiac surgery
• cardiopulmonary bypass

• Adrenal Cortex/metabolism
• Adrenal Cortex Diseases/etiology
• Adrenal Cortex Diseases/metabolism
• Animals
• Cardiopulmonary Bypass/adverse effects
• Case-Control Studies
• Cytokines/metabolism
• Disease Models, Animal
• Dogs
• Female
• Hydrocortisone/metabolism
• Inflammasomes/metabolism
• Inflammation/etiology
• Inflammation/metabolism
• Male
• NLR Family, Pyrin Domain-Containing 3 Protein/metabolism

• Gadd45β
• apoptosis
• cardiomyocyte
• ischaemia–reperfusion
• miR-206

• Animals
• Antigens, Differentiation/genetics
• Antigens, Differentiation/metabolism
• Disease Models, Animal
• Male
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Myocardial Reperfusion Injury/genetics
• Myocardial Reperfusion Injury/prevention & control
• Rats
• Rats, Sprague-Dawley

Cardiopulmonary bypass (CPB) induces inflammatory responses, and effective endogenous homeostasis is important for preventing systemic inflammation. We assessed whether plasma exosomal microRNAs in patients undergoing cardiac surgery with CPB are involved in the regulation of inflammatory responses. Plasma samples were isolated from CPB patients (n = 21) at 5 specified time points: pre-surgery, pre-CPB and 2 hours (h), 4 h and 24 h after CPB began. Plasma TNF-α expression was increased after CPB began compared to that in the pre-surgery samples. Plasma IL-8 and IL-6 expression peaked at 4 h after CPB began but was downregulated at 24 h. The number of plasma exosomes collected at 2 h (55.1 ± 8.3%), 4 h (63.8 ± 10.1%) and 24 h (83.5 ± 3.72%) after CPB began was significantly increased compared to that in the pre-CPB samples (42.8 ± 0.11%). These exosomes had a predominantly parental cellular origin from RBCs and platelets. Additionally, the plasma exosomal miR-223 levels were significantly increased after CPB began compared to those in the pre-CPB samples. Further, exosomal miR-223 from plasma collected after CPB began downregulated IL-6 and NLRP3 expression in the monocytes. Here, we present the novel findings that increased plasma exosomal miR-223 expression during cardiac surgery with CPB might play homeostatic roles in downregulating inflammatory responses through intercellular communication.

MicroRNAs (miRNA) are varied in length, under 25 nucleotides, single-stranded noncoding RNA that regulate post-transcriptional gene expression via translational repression or mRNA degradation. Elevated levels of miRNAs can be detected in systemic circulation after tissue injury, suggesting that miRNAs are released following cellular damage. Because of their remarkable stability, ease of detection in biofluids, and tissue specific expression patterns, miRNAs have the potential to be specific biomarkers of organ injury. The identification of miRNA biomarkers requires a systematic approach: 1) determine the miRNA tissue expression profiles within a mammalian species via next generation sequencing; 2) identify enriched and/or specific miRNA expression within organs of toxicologic interest, and 3) in vivo validation with tissue-specific toxicants. While miRNA tissue expression has been reported in rodents and humans, little data exists on miRNA tissue expression in the dog, a relevant toxicology species. The generation and evaluation of the first dog miRNA tissue atlas is described here.

Analysis of 16 tissues from five male beagle dogs identified 106 tissue enriched miRNAs, 60 of which were highly enriched in a single organ, and thus may serve as biomarkers of organ injury. A proof of concept study in dogs dosed with hepatotoxicants evaluated a qPCR panel of 15 tissue enriched miRNAs specific to liver, heart, skeletal muscle, pancreas, testes, and brain. Dogs with elevated serum levels of miR-122 and miR-885 had a correlative increase of alanine aminotransferase, and microscopic analysis confirmed liver damage. Other non-liver enriched miRNAs included in the screening panel were unaffected. Eli Lilly authors created a complimentary Sprague Dawely rat miRNA tissue atlas and demonstrated increased pancreas enriched miRNA levels in circulation, following caerulein administration in rat and dog.

The dog miRNA tissue atlas provides a resource for biomarker discovery and can be further mined with refinement of dog genome annotation. The 60 highly enriched tissue miRNAs identified within the dog miRNA tissue atlas could serve as diagnostic biomarkers and will require further validation by in vivo correlation to histopathology. Once validated, these tissue enriched miRNAs could be combined into a powerful qPCR screening panel to identify organ toxicity during early drug development.

The online version of this article (doi:10.1186/s12864-016-2958-x) contains supplementary material, which is available to authorized users.

There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) from BO to AO, with 68 up-regulated and 62 down-regulated. 4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs (|log2(fold-change)|>1.0, P < 0.01). Functional analysis revealed several pathways that were associated with puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans.

• Animals
• Biomarkers/blood
• Chickens/genetics
• Down-Regulation/genetics
• Gene Expression Profiling/methods
• High-Throughput Nucleotide Sequencing
• MicroRNAs/blood
• MicroRNAs/genetics
• Puberty/genetics
• Sexual Maturation/genetics
• Up-Regulation/genetics

• National Natural Science Foundation of China
• Agricultural science and technology innovation project of Jiangsu Province
• Agricultural breed, technology and model innovation of Jiangsu Province

• CABG
• Cardiac damage
• Off-pomp
• Uric acid
• miR-126
• miR-499

• mRNA
• microRNA
• myocardial infarction
• sequencing

AIM: To perform a profiling analysis of changes in intestinal microRNA (miRNA) expression during hypothermic circulatory arrest (HCA).

METHODS: A total of eight piglets were randomly divided into HCA and sham operation (SO) groups. Under general anesthesia, swine in the HCA group were subjected to hypothermic cardiopulmonary bypass at 24 °C followed by 80 min of circulatory arrest, and the reperfusion lasted for 180 min after cross-clamp removal. The counterparts in the SO group were only subjected to median sternotomy. Histopathological analysis was used to detect mucosal injury, and Pick-and-Mix custom miRNA real-time polymerase chain reaction (PCR) panels containing 306 unique primer sets were utilized to assay unpooled intestinal samples harvested from the two groups.

RESULTS: The intestinal mucosa of the animals that were subjected to 24 °C HCA exhibited representative ischemic reperfusion injury of grade 2 or 3 according to the Chiu score. Such intestinal mucosal injuries, with the subepithelial space and epithelial layer lifting away from the lamina propria, were accompanied by shortened and irregular villi. On the contrary, the intestinal mucosa remained normal in the sham-operated animals. In total, twenty-five miRNAs were differentially expressed between the two groups (15 upregulated and 10 downregulated in the HCA group). Among these, eight miRNAs (miR-122, miR-221-5p, miR-31, miR-421-5p, miR-4333, miR-499-3p, miR-542 and let-7d-3p) were significantly dysregulated (four higher and four lower). The expression of miR-122 was significantly (5.37-fold) increased in the HCA group vs the SO group, indicating that it may play a key role in HCA-induced mucosal injury.

CONCLUSION: Exposure to HCA caused intestinal miRNA dysregulation and barrier dysfunction in swine. These altered miRNAs might be related to the protection or destruction of the intestinal barrier.

• Reperfusion injury
• Cardiopulmonary bypass
• Animal model
• Barrier function
• Randomized controlled trial
• MicroRNA

• Animals
• Disease Models, Animal
• Gene Expression Profiling/methods
• Genetic Markers
• Heart Arrest, Induced/adverse effects
• Hypothermia, Induced/adverse effects
• Intestinal Mucosa/blood supply
• Intestinal Mucosa/metabolism
• Intestinal Mucosa/pathology
• Mesenteric Ischemia/etiology
• Mesenteric Ischemia/genetics
• Mesenteric Ischemia/metabolism
• Mesenteric Ischemia/pathology
• MicroRNAs/metabolism
• Permeability
• Real-Time Polymerase Chain Reaction
• Reperfusion Injury/etiology
• Reperfusion Injury/genetics
• Reperfusion Injury/metabolism
• Reperfusion Injury/pathology
• Swine

MicroRNAs (miRNAs) regulate a lot of physiological and pathological processes, including myocardial ischemia/reperfusion. Recent studies reported that knockdown of miR-92a could attenuate ischemia/reperfusion-induced myocardial injury. In the present study, we examined the potential anti-apoptotic effects of miR-92a in a rat myocardiocyte cell line, and the possible role of Smad7 in such actions.

In a preliminary bioinformatic analysis, we identified SMAD family member 7 (Smad7) as a potential target for miR-92a. A luciferase reporter assay indeed demonstrated that miR-92a could inhibit Smad7 expression. Myocardial ischemia/reperfusion was simulated in rat H9c2 cells with 24-h hypoxia followed by 12-h reoxygenation. Prior to hypoxia/reoxygenation, cells were transfected by miR-92a inhibitor. In some experiments, cells were co-transfected with siRNA-Smad7. The miR-92a inhibitor dramatically reduced the release of lactate dehydrogenase and malonaldehyde, and attenuated cardiomyocyte apoptosis. The miR-92a inhibitor increased SMAD7 protein level and decreased nuclear NF-κB p65 protein. Effects of the miR-92a inhibitor were attenuated by co-transfection with siRNA-Smad7.

Inhibiting miR-92a can attenuate myocardiocyte apoptosis induced by hypoxia/reoxygenation by targeting Smad7.

• Animals
• Apoptosis/drug effects
• Cell Hypoxia
• Cell Line
• Gene Expression Regulation
• L-Lactate Dehydrogenase/metabolism
• Malondialdehyde/metabolism
• MicroRNAs/antagonists & inhibitors
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Models, Biological
• Molecular Mimicry
• Myocardial Reperfusion Injury/genetics
• Myocardial Reperfusion Injury/metabolism
• Myocardial Reperfusion Injury/pathology
• Myocytes, Cardiac/drug effects
• Myocytes, Cardiac/metabolism
• Myocytes, Cardiac/pathology
• Oligonucleotides/pharmacology
• Oxygen/pharmacology
• RNA, Small Interfering/genetics
• RNA, Small Interfering/metabolism
• Rats
• Signal Transduction
• Smad7 Protein/antagonists & inhibitors
• Smad7 Protein/genetics
• Smad7 Protein/metabolism
• Transcription Factor RelA/antagonists & inhibitors
• Transcription Factor RelA/genetics
• Transcription Factor RelA/metabolism

Structural changes of the left and right atria associated with atrial fibrillation (AF) in mitral stenosis (MS) patients are well known, and alterations in microRNA (miRNA) expression profiles of the right atria have also been investigated. However, miRNA changes in the left atria still require delineation. This study evaluated alterations in miRNA expression profiles of left atrial tissues from MS patients with AF relative to those with normal sinus rhythm (NSR).

Sample tissues from left atrial appendages were obtained from 12 MS patients (6 with AF) during mitral valve replacement surgery. From these tissues, miRNA expression profiles were created and analyzed using a human miRNA microarray. Results were validated via reverse-transcription and quantitative PCR for 5 selected miRNAs. Potential miRNA targets were predicted and their functions and potential pathways analyzed via the miRFocus database.

The expression levels of 22 miRNAs differed between the AF and NSR groups. Relative to NSR patients, in those with AF the expression levels of 45% (10/22) of these miRNAs were significantly higher, while those of the balance (55%, 12/22) were significantly lower. Potential miRNA targets and molecular pathways were identified.

AF alters the miRNA expression profiles of the left atria of MS patients. These findings may be useful for the biological understanding of AF in MS patients.

Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. Several studies have demonstrated that microRNAs (miRNAs) are vital regulators of signalling pathways involved in I/R injury. The present study aims to quantify the altered expression levels of miRNA and mRNA upon I/R injury in a mouse heart transplantation model, and to investigate whether these miRNA can regulate genes involved in I/R injury. We performed heterotopic heart transplantation on mouse models to generate heart tissue samples with I/R and non-I/R (control). The expression levels of miRNAs as well as genes were measured in heart grafts by microarray and real time RT-PCR. miRNA alteration in cardiomyocytes exposed to hypoxia was also detected by qRT-PCR. We observed significant alterations in miRNA and gene expression profile after I/R injury. There were 39 miRNAs significantly downregulated and 20 upregulated up to 1.5 fold in heart grafts with I/R injury compared with the grafts without I/R. 48 genes were observed with 3 fold change and p<0.05 and 18 signalling pathways were enriched using Keggs pathway library. Additionally, hypoxia/reperfusion induced primary cardiomyocyte apoptosis and altered miRNA expression profiles. In conclusion, this is the first report on miRNA expression profile for heart transplantation associated with I/R injury. These findings provide us with an insight into the role of miRNA in I/R injury in heart transplantation.

• Animals
• Blotting, Western
• Cells, Cultured
• Flow Cytometry
• Heart Transplantation/adverse effects
• In Situ Nick-End Labeling
• Mice
• Mice, Inbred C57BL
• MicroRNAs/genetics
• Myocytes, Cardiac/metabolism
• RNA, Messenger/genetics
• Reperfusion Injury/genetics
• Reverse Transcriptase Polymerase Chain Reaction