In vivo evaluation of safety and performance of a tapered nitinol venous stent with inclined proximal end in an ovine iliac venous model.

A tapered stent with inclined proximal end is designed for fitting the iliac anatomically. The aim of the present study was to evaluate the safety and performance of the new stent in ovine left iliac veins. The experiment was performed in 30 adult sheep, and one nitinol-based VENA-BT® iliac venous stent (KYD stent) was implanted into each animal's left common iliac vein. Follow-up in all sheep consisted of angiographic, macroscopic, and microscopic examinations at Day 0 (< 24 h), Day 30, Day 90, Day 180 and Day 360 post-stenting (six animals per each time-point). 30 healthy ~ 50 kg sheep were included in this study and randomly divided into five groups according to the follow-up timepoint. All stents were implanted successfully into the left ovine common iliac vein. No significant migration occurred at follow-up. There is no statistically significant difference between the groups (p > 0.05), indicating no serious lumen loss occurred during the follow-up period. Common iliac venous pressure was further measured and the results further indicated the lumen patency at follow-up. Histological examinations indicated that no vessel injury and wall rupture, stent damage, and luminal thrombus occurred. There was moderate inflammatory cell infiltration around the stent in Day-0 and Day-30 groups with the average inflammation score of 2.278 and 2.167, respectively. The inflammatory reaction was significantly reduced in Day-90, Day-180 and Day-360 groups and the average inflammation scores were 0.9444 (p < 0.001, Day-90 vs Day-0), 1.167 (p < 0.001, Day-180 vs Day-0) and 0.667 (p < 0.001, Day-90 vs Day-0), respectively. The microscopic examinations found that the stents were well covered by endothelial cells in all follow-up time points. The results suggested that the KYD stent is feasible and safe in animal model. Future clinical studies may be required to further evaluate its safety and efficacy.

Identification and Validation of Hub Genes in the Stenosis of Arteriovenous Fistula.

Arteriovenous fistula (AVF) is the most widely used hemodialysis vascular access in China. However, stenosis of the AVF limits its use. The mechanism of AVF stenosis is currently unknown. Therefore, the purpose of our study was to explore the mechanisms of AVF stenosis. In this study, we identified the differentially expressed genes (DEGs) based on the Gene Expression Omnibus (GEO) dataset (GSE39488) between venous segments of AVF and normal veins. A protein-protein interaction (PPI) network was constructed to identify hub genes of AVF stenosis. Finally, six hub genes (FOS, NR4A2, EGR2, CXCR4, ATF3, and SERPINE1) were found. Combined with the results of the PPI network analysis and literature search, FOS and NR4A2 were selected as the target genes for further investigation. We validated the bioinformatic results via reverse transcription PCR (RT-PCR) and Western blot analyses on human and rat samples. The expression levels of the mRNA and protein of FOS and NR4A2 were upregulated in both human and rat samples. In summary, we found that FOS may play an important role in AVF stenosis, which could be a potential therapeutic target of AVF stenosis.

Identification of key microRNAs in exosomes derived from patients with the severe acute pancreatitis.

OBJECTIVE: Exosomes have been identified as important carriers of various genetic materials, including microRNAs (miRNAs). Increasing evidence indicates that the course of severe acute pancreatitis (SAP) is associated with miRNAs transported by exosomes. We aimed to identify the signature miRNAs as biomarkers of SAP. METHODS: We obtained exosomes from the SAP patients' blood. After separation, purification, and identification, we performed high-throughput sequencing and screened the differentially expressed(DE) miRNAs in the exosomes. Bioinformatics analysis was performed to identified the target genes of the miRNAs and the pathways enriched based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and selected the key miRNAs related to SAP. Total RNA was extracted from patient serum exosomes to detect the expression levels of the selected miRNAs in exosomes of three experimental groups (mild -, moderately severe -, and severe AP) and a control group, using Real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: 272 DE miRNAs were identified between SAP and control group. Using bioinformatics analysis, we determined that the functions of the target genes were enriched in six signaling pathways including focal adhesion. Based on this, seven candidate signature miRNAs were selected: miR-603, miR-548ad-5p, miR-122-5p, miR-4477a, miR-192-5p, miR-215-5p, and miR-583. The RT-qPCR results of the seven miRNAs in the SAP group were consistent with the sequencing results. CONCLUSION: Exosome-derived miR-603, miR-548ad-5p, miR-122-5p, miR-4477a, miR-192-5p, miR-215-5p, miR-583 are positively correlated with SAP, which might provide new insights into the pathogenesis of SAP and serve as the biomarkers of SAP.

Feasibility and safety of trans-biliary cryoablation: Preclinical evaluation of a novel flexible cryoprobe.

Cryoablation, as a well-characterized technology, has multifarious clinical applications in solid malignancy. However, trans-biliary cryoablation for malignant biliary obstruction has not been reported yet. Thus, this study aimed to determine the efficacy and safety of trans-biliary cryoablation with a novel CO2 gas-based flexible cryoprobe in standardized preclinical settings. For fresh porcine liver ex vivo, the freezing efficacy of cryoablation was evaluated by using fresh porcine liver. The real-time CO2 flow rate, freezing temperature and freezing range were examined and the frozen appearance was visualized. In vivo study, acute and chronical effects were investigated by using the models of canine bile duct. Histopathology and laboratory examination were performed. The lowest temperature that the electrode could deliver to the tissue was -60.7 °C. At 60s after freezing, the tissue temperature dropped to -22.6 °C and -4.3 °C at 0.1 and 0.2 cm from the electrode center, respectively. The frozen size was greater in liver tissue ex vivo than that in bile duct tissue in vivo. No biliary hemorrhage, perforation, stricture, obstruction, and adjacent organ injury were observed. With histopathologic examination, acute intercellular vacuoles were observed in the lamina propria adjacent to the lumen. Chronic changes, including uneven coagulative necrosis, fibro-proliferation, inflammatory infiltration and connective tissue thickening were observed in the lamina propria of the all biliary samples. The results demonstrated CO2 gas-based trans-biliary cryoablation is safe and efficacious. These findings may provide a potential new modality for primary malignant biliary obstruction and malignant obstruction within a biliary stent and contribute to cryoablation of clinical practice.

Prognostic biomarkers of pancreatic cancer identified based on a competing endogenous RNA regulatory network.

Background: Pancreatic cancer is an insidious and heterogeneous malignancy with poor prognosis that is often locally unresectable. Therefore, determining the underlying mechanisms and effective prognostic indicators of pancreatic cancer may help optimize clinical management. This study was conducted to develop a prognostic model for pancreatic cancer based on a competing endogenous RNA (ceRNA) network. Methods: We obtained transcriptomic data and corresponding clinicopathological information of pancreatic cancer samples from The Cancer Genome Atlas (TCGA) database (training set). Based on the ceRNA interaction network, we screened candidate genes to build prediction models. Univariate Cox regression analysis was performed to screen for genes associated with prognosis, and least absolute shrinkage and selection operator (LASSO) regression analysis was conducted to construct a predictive model. A receiver operating characteristic (ROC) curve was drawn, and the C-index was calculated to evaluate the accuracy of the prediction model. Furthermore, we downloaded transcriptomic data and related clinical information of pancreatic cancer samples from the Gene Expression Omnibus database (validation set) to evaluate the robustness of our prediction model. Results: Eight genes (ANLN, FHDC1, LY6D, SMAD6, ACKR4, RAB27B, AUNIP, and GPRIN3) were used to construct the prediction model, which was confirmed as an independent predictor for evaluating the prognosis of patients with pancreatic cancer through univariate and multivariate Cox regression analysis. By plotting the decision curve, we found that the risk score model is an independent predictor has the greatest impact on survival compared to pathological stage and targeted molecular therapy. Conclusions: An eight-gene prediction model was constructed for effectively and independently predicting the prognosis of patients with pancreatic cancer. These eight genes identified show potential as diagnostic and therapeutic targets.

Targeting Oxidative Stress and Inflammatory Response for Blood-Brain Barrier Protection in Intracerebral Hemorrhage.

Significance: Blood-brain barrier (BBB) disruption is a major pathological change after intracerebral hemorrhage (ICH) and is both the cause and result of oxidative stress and of the immune response post-ICH. These processes contribute to ICH-induced brain injury. Recent Advances: After the breakdown of cerebral vessels, blood components, including erythrocytes and their metabolites, thrombin, and fibrinogen, can access the cerebral parenchyma through the compromised BBB, triggering oxidative stress and inflammatory cascades. These aggravate BBB disruption and contribute to further infiltration of blood components, resulting in a vicious cycle that exacerbates brain edema and neurological injury after ICH. Experimental and clinical studies have highlighted the role of BBB disruption in ICH-induced brain injury. Critical Issues: In this review, we focus on the strategies to protect the BBB in ICH. Specifically, we summarize the evidence and the underlying mechanisms, including the ICH-induced process of oxidative stress and inflammatory response, and we highlight the potential therapeutic targets to protect BBB integrity after ICH. Future Directions: Future studies should probe the mechanism of ferroptosis as well as oxidative stress-inflammation coupling in BBB disruption after ICH and investigate the effects of antioxidants and immunomodulatory agents in more ICH clinical trials. Antioxid. Redox Signal. 37, 115-134.

ADP-ribosylation of histone variant H2AX promotes base excision repair.

Optimal DNA damage response is associated with ADP-ribosylation of histones. However, the underlying molecular mechanism of DNA damage-induced histone ADP-ribosylation remains elusive. Herein, using unbiased mass spectrometry, we identify that glutamate residue 141 (E141) of variant histone H2AX is ADP-ribosylated following oxidative DNA damage. In-depth studies performed with wild-type H2AX and the ADP-ribosylation-deficient E141A mutant suggest that H2AX ADP-ribosylation plays a critical role in base excision repair (BER). Mechanistically, ADP-ribosylation on E141 mediates the recruitment of Neil3 glycosylase to the sites of DNA damage for BER. Moreover, loss of this ADP-ribosylation enhances serine-139 phosphorylation of H2AX (γH2AX) upon oxidative DNA damage and erroneously causes the accumulation of DNA double-strand break (DSB) response factors. Taken together, these results reveal that H2AX ADP-ribosylation not only facilitates BER repair, but also suppresses the γH2AX-mediated DSB response.

NADP+ is an endogenous PARP inhibitor in DNA damage response and tumor suppression.

ADP-ribosylation is a unique posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) using NAD+ as ADP-ribose donor. PARPs play an indispensable role in DNA damage repair and small molecule PARP inhibitors have emerged as potent anticancer drugs. However, to date, PARP inhibitor treatment has been restricted to patients with BRCA1/2 mutation-associated breast and ovarian cancer. One of the major challenges to extend the therapeutic potential of PARP inhibitors to other cancer types is the absence of predictive biomarkers. Here, we show that ovarian cancer cells with higher level of NADP+, an NAD+ derivative, are more sensitive to PARP inhibitors. We demonstrate that NADP+ acts as a negative regulator and suppresses ADP-ribosylation both in vitro and in vivo. NADP+ impairs ADP-ribosylation-dependent DNA damage repair and sensitizes tumor cell to chemically synthesized PARP inhibitors. Taken together, our study identifies NADP+ as an endogenous PARP inhibitor that may have implications in cancer treatment.

Poly(ADP-Ribose) Mediates the BRCA2-Dependent Early DNA Damage Response.

Breast cancer susceptibility gene 2 (BRCA2) plays a key role in DNA damage repair for maintaining genomic stability. Previous studies have shown that BRCA2 contains three tandem oligonucleotide/oligosaccharide binding folds (OB-folds) that are involved in DNA binding during DNA double-strand break repair. However, the molecular mechanism of BRCA2 in DNA damage repair remains elusive. Unexpectedly, we found that the OB-folds of BRCA2 recognize poly(ADP-ribose) (PAR) and mediate the fast recruitment of BRCA2 to DNA lesions, which is suppressed by PARP inhibitor treatment. Cancer-associated mutations in the OB-folds of BRCA2 disrupt the interaction with PAR and abolish the fast relocation of BRCA2 to DNA lesions. The quickly recruited BRCA2 is important for the early recruitment of exonuclease 1(EXO1) and is involved in DNA end resection, the first step of homologous recombination (HR). Thus, these findings uncover a molecular mechanism by which BRCA2 participates in DNA damage repair.

The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.

The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression.

The G9a/GLP complex mediates mono- and dimethylation of Lys9 of histone H3 at specific gene loci, which is associated with transcriptional repression. However, the molecular mechanism by which the G9a/GLP complex is targeted to the specific gene loci for H3K9 methylation is unclear. In this study, with unbiased protein affinity purification, we found ZNF644 and WIZ as two core subunits in the G9a/GLP complex. ZNF644 and WIZ interact with the transcription activation domain of G9a and GLP, respectively. Moreover, both ZNF644 and WIZ contain multiple zinc finger motifs that recognize consensus DNA sequences. ZNF644 and WIZ target G9a and GLP to the chromatin and mediate the G9a/GLP complex-dependent H3K9 methylation as well as gene repression. Thus, our studies reveal two key subunits in the G9a/GLP complex that regulate the function of this histone methyltransferase complex.

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage.

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis.

Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

Crosstalk between TGF-ß/Smad3 and BMP/BMPR2 signaling pathways via miR-17-92 cluster in carotid artery restenosis.

In the recent decades, carotid angioplasty and stenting (CAS) has been developed into a credible option for the patients with carotid stenosis. However, restenosis remains a severe and unsolved issue after CAS treatment. Restenosis is characterized by neointimal hyperplasia, which is partially caused by vascular smooth muscle cells (VSMC) proliferation. However, the molecular mechanism involved in the restenosis is still unclear. In this study, we demonstrated a functional crosstalk between two TGF-ß superfamily signaling pathway members, Smad3 and BMPR2, in VSMC proliferation. Smad3 plays an important role in the TGF-ß/Smad3 signaling pathway, and is significantly upregulated in the carotid artery with restenosis to promote VSMC proliferation. In contrast, BMP receptor II (BMPR2), an inhibitor of VSMC proliferation is downregulated in carotid restenosis. We further found that BMPR2 downregulation is mediated by miR-17-92 cluster, which is transcriptionally regulated by Smad3. Thus, Smad3 upregulation and Smad3/miR-17-92 cluster-dependent BMPR2 downregulation are likely to promote VSMC proliferation and restenosis. Taken together, our results may provide novel clues for early diagnosis of carotid restenosis and developing new therapeutic strategy.

PGC7 suppresses TET3 for protecting DNA methylation.

Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

Uhrf2 is important for DNA damage response in vascular smooth muscle cells.

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.

TET2 promotes histone O-GlcNAcylation during gene transcription.

Ten eleven translocation (TET) enzymes, including TET1, TET2 and TET3, convert 5-methylcytosine to 5-hydroxymethylcytosine and regulate gene transcription. However, the molecular mechanism by which TET family enzymes regulate gene transcription remains elusive. Using protein affinity purification, here we search for functional partners of TET proteins, and find that TET2 and TET3 associate with O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), an enzyme that by itself catalyses the addition of O-GlcNAc onto serine and threonine residues (O-GlcNAcylation) in vivo. TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo. Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O-GlcNAcylation. Moreover, OGT associates with TET2 at transcription start sites. Downregulation of TET2 reduces the amount of histone 2B Ser 112 GlcNAc marks in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a TET2-dependent O-GlcNAcylation of chromatin. The double epigenetic modifications on both DNA and histones by TET2 and OGT coordinate together for the regulation of gene transcription.

miR-21 modulates the ERK-MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation.

The ERK-MAPK signaling pathway plays a pivotal role during mesenchymal stem cell (MSC) differentiation. Studies have demonstrated that ERK-MAPK promotes adipogenesis and osteogenesis through the phosphorylation of differentiation-associated transcription factors and that it is the only active signaling in all three lineages (adipogenic, chondrogenic, and osteogenic) during MSC differentiation. Recent studies pointed to the significant roles of microRNA-21 (miR-21) during several physiological and pathological processes, especially stem cell fate determination. The miR-21 expression pattern is also correlated with ERK-MAPK activity. Here, we found that miR-21 expression is elevated and associated with an increased differentiation potential in MSCs during adipogenesis and osteogenesis. The overexpression of miR-21 elevated the expression level of the differentiation-associated genes PPARγ and Cbfa-1 during MSC differentiation, whereas miR-21 knockdown reduced the expression level of both genes. The ERK-MAPK signaling pathway activity had an increasing tendency to respond to miR-21 upregulation and a decreasing tendency to respond to miR-21 down-regulation during the first 4 days of adipogenesis and osteogenesis. Our data indicate that miR-21 modulated ERK-MAPK signaling activity by repressing SPRY2 expression, a known regulator of the receptor tyrosine kinase (RTK) signaling pathway, to affect the duration and magnitude of ERK-MAPK activity. The ERK-MAPK signaling pathway was regulated by Sprouty2 (SPRY2) expression via a miR-21-mediated mechanism during MSC differentiation.

Loss of BRCA1-A complex function in RAP80 null tumor cells.

Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.