Natural recombination of the torque teno canis virus within the ORF1, -2, and -3 genes and the untranslated region.

The torque teno canis virus (TTCaV) was first reported in 2001 and it shares similarities with the known Torque teno virus (TTV) in terms of genomic organization and putative transcriptional features. It is a single-stranded DNA virus characterized by high rates of recombination and nucleotide substitution, like RNA viruses. Studies reported recombination events in torque teno virus; however, there is limited reporting of TTCaV reorganization events. This study screened fecal samples from domestic dogs in Henan Province. There was a positivity rate of 16.5% (19/115) for TTCaV. Four nearly complete TTCaV genomes, namely Canine/HeNan/4, 5, 6, and 13/2019, were obtained from the 19 positive fecal samples, whose genome sequence was obtained using gap-filling PCR. Sequence analysis revealed two unique amino acid mutation sites in the TTCaV strains, K278Q (compared with the first isolate Cf-TTV10 in Japan) and V/L268I (compared with the TTCaV strain from southern China). Subsequently, 17 near full-length TTCaV genome sequences were subjected to phylogenetic and recombination detection program analyzes. We obtained evidence supporting recombination events in the Chinese TTCaV strains. These findings suggest that mutation and recombination occurred in the three individual gene segments (ORF1, ORF2, ORF3) and the untranslated region, an area of major recombination in the Chinese TTCaV strain GX265 genome. Interestingly, the TTCaV strain (Canine/HeNan/6/2019) was a major parent involved in the genetic recombination of the GX265 strain. This study provides insights into the genetic variability and evolution of TTCaV.

Identification of Two Isoforms of Canine Tetherin in Domestic Dogs and Characterization of Their Antiviral Activity against Canine Influenza Virus.

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT-PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release.

Reactive oxygen species-induced long intergenic noncoding RNA p21 accelerates abdominal aortic aneurysm formation by promoting secretary smooth muscle cell phenotypes.

Whether long noncoding RNAs participate in the formation of abdominal aortic aneurysms (AAAs) through the regulation of SMC phenotypic switching is unknown. lincRNA-p21 induced by reactive oxygen species (ROS) is likely functionally associated with SMC phenotypic switching. We thus investigated the role of lincRNA-p21 in SMC phenotypic switching-associated AAA formation and its underlying mechanisms. An analysis of human and mouse abdominal aortic samples revealed that the lincRNA-p21 levels were significantly higher in AAA tissue. Stimulation with hydrogen peroxide upregulated the expression of lincRNA-p21 in a dose-dependent manner and converted SMCs from a contractile phenotype to a synthetic, proteolytic, and proinflammatory phenotype in vitro. Moreover, lincRNA-p21 promoted fracture of elastic fibres, reconstruction of the vascular wall, and AAA formation in vivo by modulating SMC phenotypic switching in two mouse models of AAA induced by angiotensin II or porcine pancreatic elastase (PPE) perfusion. Using a bioinformatics prediction method and luciferase reporter gene assays, we further proved that lincRNA-p21 sponged miR-204-5p to release the transcriptional activity of Mekk3 and promoted the NF-κB pathway and thereby played a role in the SMC phenotypic switch and AAA formation. The ROS levels were positively correlated with the lincRNA-p21 levels in human and mouse AAA tissues. The knockdown of lincRNA-p21 in a PPE-induced mouse AAA model increased the miR-204-5p levels and reduced the expression of Mekk3, whereas lincRNA-p21 overexpression had the opposite effect. Collectively, the results indicated that ROS-induced lincRNA-p21 sponges miR-204-5p to accelerate synthetic and proinflammatory SMC phenotypes through the Mekk3/NF-κB pathway in AAA formation. Thus, lincRNA-p21 may have therapeutic potential for AAA formation.

Genetic characterization of three recently discovered parvoviruses circulating in equines in China.

The family Parvoviridae comprises many major viral pathogens that can infect humans and multiple other species, causing severe diseases. However, knowledge of parvoviruses that infect equids is limited. In the present study, we found that three equine parvoviruses (EqPVs), namely, equine parvovirus-hepatitis (EqPV-H), equine parvovirus-cerebrospinal fluid (EqPV-CSF) and equine copivirus (EqCoPV) cocirculated among horses in China. We examined the prevalence of these three EqPVs in 225 horse serum samples in China and found EqPV-H, EqPV-CSF and EqCoPV viremia in 7.6% (17/225), 2.7% (6/225) and 2.2% of samples (5/225), respectively. We also obtained the complete genomes of one EqPV-H strain, six EqPV-CSF strains and one EqCoPV strain. After phylogenetic analysis of the EqPVs, we found that EqPV-CSF and EqCoPV may have evolved from the same ancestor. The EqPV-CSF strains (E111 and A27) and EqCoPV strain (F124) were genetically similar to foreign strains, but the EqPV-CSF strains (B48, E96, C61 and F146) comprised unique clades. This study determined the prevalence of three EqPVs in Chinese horses and analyzed the genetic characteristics of EqPVs prevalent strains in Chinese horse herds. Our data provide a theoretical basis for follow-up research on the prevention and control of EqPVs.

Identification and genetic characterization of bovine hepacivirus in China: A large scale epidemiological study.

Bovine hepacivirus (BovHepV) is a novel virus that was recently discovered in Ghana and Germany in 2015. Until now, this virus has been identified in cattle population worldwide and is classified into subtypes A-G. To fully understand the epidemic situation and genetic characteristic of BovHepV in China, a total of 612 cattle serum samples were collected from 20 farms in seven provinces and municipality in China between 2018 and 2020 and were tested for the presence of BovHepV RNA via semi-nested PCR. The results demonstrated that 49 (8.0%) samples were BovHepV RNA-positive. It is noted that BovHepV infection in yak was confirmed for the first time. BovHepV was detected in all the seven provinces, with the positive rate ranging from 3.1% to 13.3%, which indicates a wide geographical distribution pattern of BovHepV in China. Sequencing results revealed that 5' UTR of the 49 field BovHepV strains have a nucleotide similarity of 96.3%-100% between each other and 93.9%-100% with previously reported BovHepV strains. In addition, genetic analysis identified five critical nucleotide sites in 5' UTR to distinguish different subtypes, which was further verified by genomic sequencing and nucleotide similarity analysis. All the 49 Chinese field BovHepV strains were classified into subtype G and this subtype is only determined in cattle in China currently. This study will provide insights for us to better understand the epidemiology and genetic diversity of BovHepV.

Swine Interferon-Inducible Transmembrane Proteins Potently Inhibit African Swine Fever Virus Replication.

African swine fever virus (ASFV) causes an acute, hemorrhagic, and highly contagious disease in domestic swine, leading to significant economic losses to the global porcine industry. Restriction factors of innate immunity play a critical in host antiviral action. However, function of swine restriction factors of innate immunity on ASFV has been seldomly investigated. In this study, we determined five homologues of swine interferon-induced transmembrane proteins (SwIFITM [named SwIFITM1a, -1b, -2, -3, and -5]), and we found that they all exhibit potent antiviral activity against ASFV. Expression profile analysis indicated that these SwIFITMs are constitutively expressed in most porcine tissues. Whether infected with ASFV or treated with swine interferon, the expression levels of SwIFITMs were induced in vitro. The subcellular localization of SwIFITMs was similar to that of their human homologues. SwIFITM1a and -1b localized to the plasma membrane, SwIFITM2 and -3 focused on the cytoplasm and the perinuclear region, while SwIFITM5 accumulated in the cell surface and cytoplasm. The overexpression of SwIFITM1a, -1b, -2, -3, or -5 could significantly inhibit ASFV replication in Vero cells, whereas knockdown of these genes could enhance ASFV replication in PAMs. We blocked the constitutive expression of endogenous IFITMs in Vero cells using a CRISPR-Cas9 system and then infected them with ASFV. The results indicated that the knockout of endogenous IFITMs could enhance ASFV replication. Finally, we expressed five SwIFITMs in knockout Vero cell lines and then challenged them with ASFV. The results showed that all of the SwIFITMs had a strong antiviral effect on ASFV. This research will further expand the understanding of the anti-ASFV activity of porcine IFITMs.

MicroRNA-23b prevents aortic aneurysm formation by inhibiting smooth muscle cell phenotypic switching via FoxO4 suppression.

AIMS: Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for the formation of abdominal aortic aneurysms (AAAs). MicroRNA-23b (miR-23b) has recently been shown to play a vital role in maintaining the VSMC contractile phenotype; however, little is known about the role of miR-23b in the formation of AAAs. Here, we investigated whether miR-23b prevents AAA formation by inhibiting VSMC phenotypic switching. MATERIALS AND METHODS: We administered angiotensin II (Ang II, 1000 ng/kg/min) or vehicle to 10-12-week-old male apolipoprotein E knockout (ApoE-/-) or C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks. KEY FINDINGS: The expression of miR-23b was significantly reduced in the aorta during the early onset of AAA in angiotensin II-treated ApoE-/- mice and in human AAA samples. In vitro experiments showed that the suppression of SMC contractile marker gene expression induced by Ang II was accelerated by miR-23b inhibitors but inhibited by mimics. In vivo studies revealed that miR-23b deficiency in Ang II-treated C57BL/6J mice aggravated the formation of AAAs in these mice compared with control mice; the opposite results were observed in miR-23b-overexpressing mice. Mechanistically, miR-23b knockdown significantly increased the expression of the transcription factor forkhead box O4 (FoxO4) during VSMC phenotypic switching induced by Ang II. In addition, a luciferase reporter assay showed that FoxO4 is a target of miR-23b in VSMCs. SIGNIFICANCE: Our study revealed a pivotal role for miR-23b in protecting against aortic aneurysm formation by maintaining the VSMC contractile phenotype.

Circular RNA Cdyl promotes abdominal aortic aneurysm formation by inducing M1 macrophage polarization and M1-type inflammation.

Macrophage polarization plays a crucial role in regulating abdominal aortic aneurysm (AAA) formation. Circular RNAs (circRNAs) are important regulators of macrophage polarization during the development of cardiovascular diseases. How-ever, the roles of circRNAs in regulating AAA formation through modulation of macrophage polarization remain unknown. In the present study, we compared circRNA microarray data under two distinct polarizing conditions (M1 and M2 macrophages) and identified an M1-enriched circRNA, circCdyl. Loss- and gain-of-function assay results demonstrated that circCdyl overexpression accelerated angiotensin II (Ang II)- and calcium chloride (CaCl2)-induced AAA formation by promoting M1 polarization and M1-type inflammation, while circCdyl deficiency showed the opposite effects. RNA pulldown, mass spectrometry analysis, and RNA immunoprecipitation (RIP) assays were conducted to elucidate the underlying mechanisms by which circCdyl regulates AAA formation and showed that circCdyl promotes vascular inflammation and M1 polarization by inhibiting interferon regulatory factor 4 (IRF4) entry into the nucleus, significantly inducing AAA formation. In addition, circCdyl was shown to act as a let-7c sponge, promoting C/EBP-δ expression in macrophages to induce M1 polarization. Our results indicate an important role for circCdyl-mediated macrophage polarization in AAA formation and provide a potent therapeutic target for AAA treatment.

Canine Interferon-Inducible Transmembrane Protein Is a Host Restriction Factor That Potently Inhibits Replication of Emerging Canine Influenza Virus.

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro. An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host's defense against virus infection.

Loss of exosomal LncRNA HCG15 prevents acute myocardial ischemic injury through the NF-κB/p65 and p38 pathways.

Exosomes are nanosized bilayer membrane vesicles that may mediate intercellular communication by transporting bioactive molecules, including noncoding RNAs, mRNAs, and proteins. Research has shown that exosomes play an important role in acute myocardial infarction (AMI), but the function and regulation of exosomal long noncoding RNAs (lncRNAs) in AMI are unclear. Thus, RNA sequencing (RNA-Seq) was conducted to investigate the exosomal lncRNA transcriptome from MI patients and identified 65 differentially expressed lncRNAs between the MI and control groups. HCG15, one of the differentially expressed lncRNAs, was verified to have the highest correlation with cTnT by qRT-PCR, and it also contributed to the diagnosis of AMI by receiver operating characteristic (ROC) analysis. Upregulation of HCG15 expression facilitated cardiomyocyte apoptosis and inflammatory cytokine production and inhibited cell proliferation. We also confirmed that HCG15 was mainly wrapped in exosomes from AC16 cardiomyocytes under hypoxia, which contributed to cardiomyocyte apoptosis, the release of inflammatory factors, and inhibition of cell proliferation via the activation of the NF-κB/p65 and p38 pathways, whereas suppressing the expression of HCG15exerted opposite effects, In addition, Overexpression of HCG15 aggravated cardiac IR injury in C57BL/6J mice. This study not only helps elucidate exosomal lncRNA function in AMI pathogenesis but also contributes to the development of novel therapeutic strategies.

First identification and genomic characterization of equine hepacivirus subtype 2 in China.

Equine hepacivirus (EqHV) is a newly discovered hepatitis C virus-like virus that can infect equines. EqHV strains circulating worldwide have been classified into subtypes 1-3. In previous studies, we detected the presence of EqHV strains of subtype 1 and 3 in China. To determine whether EqHV strains of subtype 2 are prevalent in China, serum samples were collected from 133 racehorses in Guangdong province in 2021 and were tested for EqHV RNA by RT-PCR, and the positive rate was 9% (12/133). Sequencing of the NS3 gene revealed that one field strain (GD2021) had a high degree of genetic similarity to EqHV strains of subtype 2. Subsequent genome sequencing and analysis demonstrated that strain GD2021 belongs to subtype 2. The present study enriches our knowledge about the genetic diversity of EqHV in China.

Novel parvovirus in cats, China.

Parvovirus is a common element of the feline virus group and usually causes gastroenteritis and leukopenia in cats. In this study, we identified a novel protoparvovirus from the Chinese domestic cats, which is genetically similar to canine bufavirus (98.0%-99.8%), but sharing low amino acid identities in the viral structural proteins 2 (VP2) (36.1-37.2%) to the well-known canine parvovirus type 2 and feline panleukopenia virus. This virus was provisionally designated as feline bufavirus (FBuV). Screening of fecal samples revealed a prevalence of 7.4% (19/257) in domestic cats. Diarrhea was present in 52.6% (10/19) of cats positive for FBuV. However, statistical analysis showed no association between FBuV and clinical signs. VP2 gene of the 19 field FBuV was sequenced and phylogenetic analysis demonstrated that FBuV determined from China had a genetic diversity. This study will strengthen the understanding of the epidemiology and genetic diversity of bufavirus and provide a foundation for further studies.

Preliminary Study on the Safety and Efficacy of One-Stop Treatment of Percutaneous LAAO Combined with Coronary Intervention for Higher Risk of Bleeding in Patients with AF Complicated with CHD.

AIMS: To explore the feasibility, safety, and efficacy of 1-stop treatment of percutaneous left atrial appendage occlusion (LAAO) combined with coronary intervention for patients with nonvalvular atrial fibrillation (AF) complicated with coronary heart disease (CHD). METHODS AND RESULTS: We retrospectively analyzed the clinical data of 6 patients with AF combined with CHD admitted from Zhuhai People's Hospital from April 2017 to June 2018. After the operation, all patients were treated with aspirin (100 mg qd) and clopidogrel (75 mg qd) for 1 year, which is considered long-term use of aspirin/clopidogrel. The effects of LAAO and coronary intervention were evaluated immediately. The location of the left atrial appendage occluder, thrombosis, residual leak, and clinical manifestations were observed during the 90-day follow-up. The patients were implanted with Watchman™ devices and coronary stents. After the operation, the immediate sealing effect was satisfactory. The Watchman occluder was used in accordance with the PASS principle (position, anchor, size, seal), and the coronary intervention was satisfactory. During the operation, there were no device-related thrombosis, tamponade, or vascular complications. Follow-up results showed that in the 6 patients, there were no hemorrhagic strokes, worsening heart function, residual leakage, device-related thrombosis, angina pectoris, myocardial infarction, skin ecchymosis, gastrointestinal bleeding, or cerebral hemorrhage. CONCLUSION: For patients with nonvalvular AF combined with CHD, the safety and feasibility of 1-stop treatment with left atrial appendage and coronary intervention are reliable, and the curative effects were also satisfactory at short- and medium-term follow-up.

Loss of FoxO3a prevents aortic aneurysm formation through maintenance of VSMC homeostasis.

Vascular smooth muscle cell (VSMC) phenotypic switching plays a critical role in the formation of abdominal aortic aneurysms (AAAs). FoxO3a is a key suppressor of VSMC homeostasis. We found that in human and animal AAA tissues, FoxO3a was upregulated, SM22α and α-smooth muscle actin (α-SMA) proteins were downregulated and synthetic phenotypic markers were upregulated, indicating that VSMC phenotypic switching occurred in these diseased tissues. In addition, in cultured VSMCs, significant enhancement of FoxO3a expression was found during angiotensin II (Ang II)-induced VSMC phenotypic switching. In vivo, FoxO3a overexpression in C57BL/6J mice treated with Ang II increased the formation of AAAs, whereas FoxO3a knockdown exerted an inhibitory effect on AAA formation in ApoE-/- mice infused with Ang II. Mechanistically, FoxO3a overexpression significantly inhibited the expression of differentiated smooth muscle cell (SMC) markers, activated autophagy, the essential repressor of VSMC homeostasis, and promoted AAA formation. Our study revealed that FoxO3a promotes VSMC phenotypic switching to accelerate AAA formation through the P62/LC3BII autophagy signaling pathway and that therapeutic approaches that decrease FoxO3a expression may prevent AAA formation.

METTL3 Induces AAA Development and Progression by Modulating N6-Methyladenosine-Dependent Primary miR34a Processing.

Identifying effective drugs to delay the progression of aortic aneurysms is a formidable challenge in vascular medicine. Methyltransferase-like 3 (METTL3) plays a key role in catalyzing the formation of N6-methyladenosine (m6A), but despite the functional importance of METTL3 and m6A in various fundamental biological processes, their roles in abdominal aortic aneurysm (AAA) are unknown. Here, we found that METTL3 knockdown in apolipoprotein E-deficient (ApoE-/-) mice treated with angiotensin II suppressed the formation of AAAs, while METTL3 overexpression exerted the opposite effects. Similar results were obtained in a calcium chloride (CaCl2)-induced mouse AAA model. Mechanistically, METTL3-dependent m6A methylation promoted primary microRNA-34a (miR-34a, pri-miR34a) maturation through DGCR8. Moreover, miR-34a overexpression significantly decreased SIRT1 expression and aggravated AAA formation, while miR-34a deficiency produced the opposite effects. In a rescue experiment, miR-34a knockdown or forced expression of SIRT1 partially attenuated the protective effects of METTL3 deficiency against AAA formation. Our studies reveal an important role for METTL3/m6A-mediated miR-34a maturation in AAA formation and provide a novel therapeutic target and diagnostic biomarker for AAA treatment.

Itaconate prevents abdominal aortic aneurysm formation through inhibiting inflammation via activation of Nrf2.

BACKGROUND: Identifying effective drugs to suppress vascular inflammation is a promising strategy to delay the progression of abdominal aortic aneurysm (AAA). Itaconate has a vital role in regulating inflammatory activation in various inflammatory diseases. However, the role of itaconate in the progression of AAA is unknown. In this study, we explored the inhibitory effect of itaconate on AAA formation and its underlying mechanisms. METHODS: Quantitative PCR, western blotting and immunohistochemistry were used to determine Irg1 and downstream Nrf2 expression in human and mouse AAA samples. Liquid chromatograph-mass spectrometry (LC-MS) analysis was performed to measure the abundance of itaconate. OI treatment and Irg1 knockdown were performed to study the role of OI in AAA formation. Nrf2 intervention in vivo was performed to detect the critical role of Nrf2 in the beneficial effect of OI on AAA. FINDINGS: We found that itaconate suppressed the formation of angiotensin II (Ang II)-induced AAA in apolipoprotein E-deficient (Apoe-/-) mice, while Irg1 deficiency exerted the opposite effect. Mechanistically, itaconate inhibited vascular inflammation by enabling Nrf2 to function as a transcriptional repressor of downstream inflammatory genes via alkylation of Keap1. Moreover, Nrf2 deficiency significantly aggravated inflammatory factor expression and promoted AAA formation. In addition, Keap1 overexpression significantly promoted Ang II-induced AAA formation, which was inhibited by itaconate. INTERPRETATION: Itaconate inhibited AAA formation by suppressing vascular inflammation, and therapeutic approaches to increase itaconate are potentially beneficial for preventing AAA formation. FUNDING: National Natural Science Foundations of China and Guangzhou regenerative medicine and Health Laboratory of Guangdong.

Alterations in gut microbiota of abdominal aortic aneurysm mice.

BACKGROUND: The gut microbiome plays an important role in various cardiovascular diseases, such as atherosclerosis and hypertension, which are associated with abdominal aortic aneurysms (AAAs). METHODS: Here, we used 16S rRNA sequencing to explore gut microbiota in C57BL ApoE-/- mice with AAAs. A mouse model of abdominal aortic aneurysms was induced with angiotensin II (Ang II) (1000 ng/min per kg). On day 28 after the operation, fecal samples were collected and stored at - 80 °C until DNA extraction. We determined the relative abundances of bacterial taxonomic groups using 16S rRNA amplicon metabarcoding, and sequences were analyzed using a combination of mother software and UPARSE. RESULTS: We found that the gut microbiome was different between control and AAA mice. The results of correlation analysis between AAA diameter and the gut microbiome as well as LEfSe of the genera Akkermansia, Odoribacter, Helicobacter and Ruminococcus might be important in the progression of AAAs. CONCLUSIONS: AAA mice is subjected to gut microbial dysbiosis, and gut microbiota might be a potential target for further investigation.

Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis.

Objective: Long noncoding RNAs (lncRNAs) may serve as specific targets for the treatment of abdominal aortic aneurysms (AAAs). LncRNA GAS5, functionally associated with smooth muscle cell (SMC) apoptosis and proliferation, is likely involved in AAA formation, but the exact role of GAS5 in AAA is unknown. We thus explored the contribution of GAS5 to SMC-regulated AAA formation and its underlying mechanisms. Methods: Human specimens were used to verify the diverse expression of GAS5 in normal and AAA tissues. The angiotensin II (Ang II)-induced AAA model in ApoE-/- mice and the CaCl2-induced AAA model in wild-type C57BL/6 mice were used. RNA pull-down and luciferase reporter gene assays were performed in human aortic SMCs to detect the interaction between GAS5 and its downstream targets of protein or microRNA (miR). Results: GAS5 expression was significantly upregulated in human AAA specimens and two murine AAA models compared to human normal aortas and murine sham-operated controls. GAS5 overexpression induced SMC apoptosis and repressed its proliferation, thereby promoting AAA formation in two murine AAA models. Y-box-binding protein 1 (YBX1) was identified as a direct target of GAS5 while it also formed a positive feedback loop with GAS5 to regulate the downstream target p21. Furthermore, GAS5 acted as a miR-21 sponge to release phosphatase and tensin homolog from repression, which blocked the activation and phosphorylation of Akt to inhibit proliferation and promote apoptosis in SMCs. Conclusion: The LncRNA GAS5 contributes to SMC survival during AAA formation. Thus, GAS5 might serve as a novel target against AAA.

The pseudogene PTENP1 regulates smooth muscle cells as a competing endogenous RNA.

The long non-coding RNA (lncRNA) PTENP1 is a pseudogene of phosphatase and tensin homologue deleted on chromosome ten (PTEN), has been implicated in smooth muscle cell (SMC) proliferation and apoptosis. PTENP1 is the pseudogene of PTEN. However, it is unclear whether and how PTENP1 functions in the proliferation and apoptosis of human aortic SMCs (HASMCs). Here, we hypothesised that PTENP1 inhibits HASMC proliferation and enhances apoptosis by promoting PTEN expression. PCR analysis and Western blot assays respectively showed that both PTENP1 and PTEN were up-regulated in human aortic dissection (AD) samples. PTENP1 overexpression significantly increased the protein expression of PTEN, promoted apoptosis and inhibited the proliferation of HASMCs. PTENP1 silencing exhibited the opposite effects and mitigated H2O2-induced apoptosis of HASMCs. In an angiotensin II (Ang II)-induced mouse aortic aneurysm (AA) model, PTENP1 overexpression potentiated aortic SMC apoptosis, exacerbated aneurysm formation. Mechanistically, RNA pull-down assay and a series of luciferase reporter assays using miR-21 mimics or inhibitors identified PTENP1 as a molecular sponge for miR-21 to endogenously compete for the binding between miR-21 and the PTEN transcript, releasing PTEN expression. This finding was further supported by in vitro immunofluorescent evidence showing decreased cell apoptosis upon miR-21 mimic administration under baseline PTENP1 overexpression. Ex vivo rescue of PTEN significantly mitigated the SMC apoptosis induced by PTENP1 overexpression. Finally, Western blot assays showed substantially reduced Akt phosphorylation and cyclin D1 and cyclin E levels with up-regulated PTENP1 in HASMCs. Our study identified PTENP1 as a mediator of HASMC homeostasis and suggests that PTENP1 is a potential target in AD or AA intervention.

LncRNA H19 promotes vascular inflammation and abdominal aortic aneurysm formation by functioning as a competing endogenous RNA.

Abdominal aortic aneurysm (AAA) is accepted as a chronic vascular inflammatory disease. However, how the inflammatory response is regulated during AAA formation is not fully understood. This study was undertaken to determine whether the long noncoding RNA (lncRNA) H19 (H19) promotes AAA formation by enhancing aortic inflammation. qRT-PCR detected the upregulation of H19 in human and mouse AAA tissue samples. Co-staining for H19 and the macrophage marker MAC-2 showed that H19 was located in vascular smooth muscle cells (VSMCs) and infiltrating aortic macrophages. In vivo overexpression of H19 increased vascular inflammation and induced AAA formation, which was supported by exacerbated aortic morphology, maximum aortic diameter values, elastin degradation, expression of interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1), and macrophage infiltration. H19 suppression resulted in the opposite effects. A rescue experiment indicated that IL-6 neutralization significantly mitigated the aortic inflammation and AAA formation evoked by H19 overexpression. Luciferase reporter assays and ex vivo experiments using VSMCs and macrophages confirmed that H19 induced aneurysm formation in part via endogenous competition with the let-7a microRNA to induce the transcription of its target gene, IL-6. This mechanism was further validated by in vivo experiments using a mutant H19 that could not effectively bind let-7a. Collectively, our study revealed a pathogenic H19/let-7a/IL-6 inflammatory pathway in AAA formation, which offers a new potential therapeutic strategy for AAA.