Treatment with the Prolyl Hydroxylase Inhibitor JNJ Promotes Abdominal Aortic Aneurysm Progression in Diabetic Mice.

OBJECTIVE: Prolyl hydroxylase domain containing proteins (PHD) rigorously regulate intracellular hypoxia inducible factor-1 (HIF-1) protein expression and activity. Diabetes impairs PHD activity and attenuates abdominal aortic aneurysm (AAA) progression. The extent to which dysregulated PHD activity contributes to diabetes mediated AAA suppression remains undetermined. METHODS: AAAs were induced in diabetic and non-diabetic male C57BL/6J mice via intra-aortic elastase infusion. A PHD inhibitor (JNJ-42041935, aka "JNJ", 150 mmol/kg) or vehicle alone was administered daily starting one day prior to AAA induction for 14 days. Influences on AAA progression was assessed via ultrasonography and histopathology. Expression of aortic HIF-1α, three of its target genes and macrophage derived mediators were assayed via quantitative reverse transcription polymerase chain reaction. Aneurysmal sections from AAA patients with and without diabetes (two patients in each group) were immunostained for HIF-1α and vascular endothelial growth factor (VEGF)-A. RESULTS: Expression of HIF-1α target genes (erythropoietin, VEGF-A, and glucose transporter-1) was reduced by 45% - 95% in experimental diabetic aortas. Diameter enlargement was similarly limited, as were mural elastin degradation, leukocyte infiltration, and neo-angiogenesis (reduced capillary density and length) on histopathology. Pre-treatment with JNJ prior to AAA initiation augmented aortic HIF-1α target gene expression and aneurysm progression in diabetic mice, along with macrophage VEGF-A and matrix metalloproteinase 2 mRNA expression. No differences were noted in HIF-1α or VEGF-A expression on aortic immunohistochemical staining of human aortic tissue as a function of diabetes status. CONCLUSION: Small molecule PHD inhibitor treatment reduces or offsets impairment of experimental AAA progression in hyperglycemic mice, highlighting the potential contribution of dysregulated PHD activity to diabetes mediated aneurysm suppression.

Electron diffraction of 1,4-dichlorobenzene embedded in superfluid helium droplets.

We perform electron diffraction of 1,4-dichlorobenzene (C6H4Cl2, referred to as 2ClB) embedded in superfluid helium droplets to investigate the structure evolution of cluster growth. Multivariable linear regression fittings are used to determine the concentration and the best model structures of the clusters. At a droplet source temperature of 22 K with droplets containing on average 5000 He atoms, the fitting results agree with the doping statistics modeled using the Poisson distribution: the largest molecular clusters are tetramers, while the abundances of monomers and dimers are the highest and are similar. Molecular dimers of 2ClB are determined to have a parallel structure with a 60° rotation for the Cl-Cl molecular axes. However, a better agreement between experiment and fitting is obtained by reducing the interlayer distance that had been calculated using the density functional theory for dimers. Further calculations using the highest level quantum mechanical calculations prove that the reduction in interlayer distance does not significantly increase the energy of the dimer. Cluster trimers adopt a dimer structure with the additional monomer slanted against the dimer, and tetramers take on a stacked structure. The structure evolution with cluster size is extraordinary, because from trimer to tetramer, one monomer needs to be rearranged, and neither the trimer nor the tetramer adopts the corresponding global minimum structure obtained using high level coupled-cluster theory calculations. This phenomenon may be related to the fast cooling process in superfluid helium droplets during cluster formation.

Inhibition of VEGF (Vascular Endothelial Growth Factor)-A or its Receptor Activity Suppresses Experimental Aneurysm Progression in the Aortic Elastase Infusion Model.

OBJECTIVE: We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad [adenovirus]-VEGFR-2), anti-VEGF-A mAb (monoclonal antibody), and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A-positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti-VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 (matrix metalloproteinase) and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice. CONCLUSIONS: VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.

Inhibition of Phosphatidylinositol 3-Kinase γ by IPI-549 Attenuates Abdominal Aortic Aneurysm Formation in Mice.

OBJECTIVE: The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway plays a pivotal role in abdominal aortic aneurysm (AAA). However, systemic inhibition of this pathway causes serious side effects, thus limiting the clinical use of pan-PI3K inhibitors. In this study, it was hypothesised that the γ subunit of PI3K plays an important role in the PI3K/AKT signalling pathway during AAA, and that specifically targeting PI3Kγ may prevent this process. METHODS: Aortic specimens were collected from AAA patients and organ donors. Furthermore, a classical AAA model in male C57BL/6 mice was created via an intra-aortic porcine pancreatic elastase (PPE) infusion and aortas were collected. A specific PI3Kγ inhibitor, IPI-549, was administered to mice orally. The protein expression level of PI3Kγ was examined by immunohistochemistry and western blotting. The aortic leukocytes were examined by immunohistochemistry and flow cytometry. RESULTS: PI3Kγ protein levels were elevated in the aortas of AAA patients and PPE infused mice. Three color immunofluorescence staining revealed the predominant area of PI3Kγ by T cells and macrophages in aneurysmal aortas. IPI-549 treatment significantly prevented AAA formation in mice. Aortic macrophages, T cells and neo-angiogenesis were significantly reduced in mice treated with IPI-549 compared with vehicle treated PPE infused mice. Flow cytometry analysis also revealed that CD45+ leukocytes and CD45+ F4/80+ macrophages in IPI-549 treated mouse aortas decreased dramatically. Additionally, IPI-549 treatment inhibited the phosphorylation of AKT in experimental aneurysmal lesions. CONCLUSION: Specific inhibition of PI3Kγ limits AAA formation. Targeting PI3Kγ prevents inflammatory cell infiltration through inhibition of AKT phosphorylation in AAA.

Hypoxia-inducible factor 1 in clinical and experimental aortic aneurysm disease.

OBJECTIVE: Mural angiogenesis and macrophage accumulation are two pathologic hallmarks of abdominal aortic aneurysm (AAA) disease. The heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) is an essential regulator of angiogenesis and macrophage function. In this study, we investigated HIF-1 expression and activity in clinical and experimental AAA disease. METHODS: Human aortic samples were obtained from 24 AAA patients and six organ donors during open abdominal surgery. Experimental AAAs were created in 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase (PPE). Expression of HIF-1α and its target gene messenger RNA (mRNA) levels were assessed in aneurysmal and control aortae. The HIF-1α inhibitors 2-methoxyestradiol and digoxin, the prolyl hydroxylase domain-containing protein (PHD) inhibitors cobalt chloride and JNJ-42041935, and the vehicle alone as control were administered daily to mice at varying time points beginning before or after PPE infusion. Influences on experimental AAA formation and progression were assessed by serial transabdominal ultrasound measurements of aortic diameter and histopathologic analysis at sacrifice. RESULTS: The mRNA levels for HIF-1α, vascular endothelial growth factor A, glucose transporter 1, and matrix metalloproteinase 2 were significantly increased in both human and experimental aneurysm tissue. Tissue immunostaining detected more HIF-1α protein in both human and experimental aneurysmal aortae compared with respective control aortae. Treatment with either HIF-1α inhibitor, beginning before or after PPE infusion, prevented enlargement of experimental aneurysms. Both HIF-1α inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and B cells. Whereas mRNA levels for PHD1 and PHD2 were elevated in experimental aneurysmal aortae, pharmacologic inhibition of PHDs had limited effect on experimental aneurysm progression. CONCLUSIONS: Expression of HIF-1α and its target genes is increased in human and experimental AAAs. Treatment with HIF-1α inhibitors limits experimental AAA progression, with histologic evidence of attenuated mural leukocyte infiltration and angiogenesis. These findings underscore the potential significance of HIF-1α in aneurysm pathogenesis and as a target for pharmacologic suppression of AAA disease.

Hypoxia-Inducible Factor 1 alpha (HIF-1α)/Vascular Endothelial Growth Factor (VEGF) Pathway Participates in Angiogenesis of Myocardial Infarction in Muscone-Treated Mice: Preliminary Study.

BACKGROUND Angiogenesis plays a crucial role in myocardial infarction (MI) treatment by ameliorating myocardial remodeling, thus improving cardiac function and preventing heart failure. Muscone has been reported to have beneficial effects on cardiac remodeling in MI mice. However, the effects of muscone on angiogenesis in MI mice and its underlying mechanisms remain unknown. MATERIAL AND METHODS Mice were randomly divided into sham, MI, and MI+muscone groups. The MI mouse model was established by ligating the left anterior descending coronary artery. Mice in the sham group received the same procedure except for ligation. Mice were administered muscone or an equivalent volume of saline for 4 consecutive weeks. Cardiac function was evaluated by echocardiograph after MI for 2 and 4 weeks. Four weeks later, all mice were sacrificed and Masson's trichrome staining was used to assess myocardial fibrosis. Isolectin B4 staining was applied to evaluate the angiogenesis in mouse hearts. Immunohistochemistry, Western blot analysis, and quantitative real-time polymerase chain reaction (qPCR) were performed to analyze expression levels of HIF-1a and its downstream genes. RESULTS Compared with the MI group, muscone treatment significantly improved cardiac function and reduced myocardial fibrosis. Moreover, muscone enhanced angiogenesis in the peri-infarct region and p-VEGFR2 expression in the vascular endothelial cells. Western blot analysis and qPCR showed that muscone upregulated expression levels of HIF-1a and VEGFA. CONCLUSIONS Muscone improved cardiac function in MI mice through augmented angiogenesis. The potential mechanism of muscone treatment in regulating angiogenesis of MI mice was upregulating expression levels of HIF-1α and VEGFA.

MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1.

BACKGROUND/AIMS: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. METHODS: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. RESULTS: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. CONCLUSIONS: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

Exogenous HGF Prevents Cardiomyocytes from Apoptosis after Hypoxia via Up-Regulating Cell Autophagy.

BACKGROUND: Hepatocyte growth factor (HGF) is widely known as a protective factor in ischemic myocardium, however HGF sensitive cellular mechanism remained ill-defined. Autophagy at early stage of hypoxia has been demonstrated to play a role in protecting myocardium both in vivo and vitro. We performed this study to investigate the association between the protective effect of HGF and autophagy. METHODS: Ventricular myocytes were isolated from neonatal rat heart (NRVMs). We evaluated cardiomyocytes apoptosis by Hoechst staining and flow cytometry. Autophagy was assessed by transmission electron microscope and mRFP-GFP-LC3 adenovirus infection. Mitochondrial membrane potential was estimated by JC-1 staining. Western blotting and ELISA assay were used to quantify protein concentrations. RESULTS: We found that autophagy in NRVMs increased at early stage after hypoxia and HGF release was consistent with the change of autophagy. Exogenous HGF enhanced autophagy and decreased apoptosis, while neutralizing HGF yielded opposite effects. Besides, inhibition of autophagy increased apoptosis of myocytes. Furthermore, exogenous HGF induced Parkin, the marker of mitochondrial autophagy, indicating increased clearance of injured mitochondria. CONCLUSIONS: Our results revealed a potential mechanism in which exogenous HGF prevented NRVMs from apoptosis after hypoxia. Upregulation of Parkin through administration of exogenous HGF may be a potential therapeutic strategy ptotecting myocytes during ischemia.

Taurine Reduced Epidural Fibrosis in Rat Models after Laminectomy via Downregulating EGR1.

BACKGROUND/AIMS: Epidural fibrosis, a common complication after laminectomy, has been demonstrated to be closely associated with poor surgical outcomes. Previous studies showed that taurine had remarkable anti-fibrotic effects on lung and liver fibrosis. We performed this study to investigate the effects of taurine in rat models of epidural fibrosis after laminectomy and to explore the potential molecular mechanism. METHODS: Laminectomy was performed on each rat to establish epidural fibrosis model. After taurine treatment, Masson's trichrome and immunohistochemistry staining were used to examine epidural fibrosis. Cell viability was determined using the Cell Counting Kit-8 assay. Annexin V/Propidium Iodide double staining was performed to detect fibroblasts apoptosis. Microarray was adopted to identify significantly changed mRNAs. mRNA expression was measured by qRT-PCR. Lentivirus infection was performed to establish stable knockdown and overexpression cell lines. The expression of fibrosis-related proteins was determined via Western blot. RESULTS: Taurine treatment markedly reduced laminectomy-induced epidural fibrosis in rat models. However, this effect of taurine was independent on TGF-ß/Smad pathway, evidenced by no change in the expression of TGF-ß and its receptors. Besides, taurine had almost no effect on cell apoptosis. Interestingly, taurine treatment significantly decreased expression of EGR1 (Early growth response protein 1), an enhancer of fibrosis, both in vivo and in vitro. Furthermore, overexpression of EGR1 increased activation of fibroblasts, while EGR1 knockdown achieved an opposite effect, indicating that EGR1 plays a key role in the inhibitory effect of taurine on TGF-ß-induced fibrosis. CONCLUSIONS: Reduced epidural fibrosis in vivo and decreased activation of fibroblasts in vitro after taurine treatment was mediated by EGR1. Taurine promises to be a potential prevention for epidural fibrosis after laminectomy.

ER Stress via CHOP Pathway is Involved in FK506-Induced Apoptosis in Rat Fibroblasts.

BACKGROUND/AIMS: Hypertrophic scars (HS) formation results from reduced apoptosis and increased proliferation of fibroblasts. Therefore, apoptosis of fibroblasts is a key target for the development of novel therapeutic strategies for HS. Previous reports demonstrated that FK506 could attenuate scar formation in vivo and FK506 could also induce endoplasmic reticulum stress (ER stress). However, the effects of FK506 on ER stress-mediated apoptosis in fibroblasts remain unclear. METHODS: Rat skin fibroblasts were used in the study. Cell viability was examined using cell counting Kit-8. Apoptosis was detected by Annexin V/Propidium Iodide Double Staining. Gene silencing was performed using Small Interfering RNAs (siRNAs) or via lentiviral infection. The expression of apoptosis-related proteins was determined via Western blot. Interaction between proteins was explored by co-immunoprecipitation. RESULTS: FK506 significantly reduced cell viability and induced apoptosis in fibroblasts. Interestingly, ER stress was also activated after FK506 treatment. We further demonstrated that FK506-induced apoptosis was mediated by ER stress via activating CHOP, evidenced by decreased apoptosis after inhibition of ER stress using TUDCA or silencing expression of CHOP. Furthermore, Co-immunoprecipitation results indicated that treatment of FK506 induced disassociation of FKBP12.6 from RyR2 and its translocation from ER membrane to cytosol, consequently promoting ER stress-mediated apoptosis. CONCLUSION: FK506-induced fibroblasts apoptosis was mediated by ER stress via CHOP signaling pathway.

Size and Site Dependence of the Catalytic Activity of Iridium Clusters toward Ethane Dehydrogenation.

This research focuses on optimizing transition metal nanocatalyst immobilization and activity to enhance ethane dehydrogenation. Ethane dehydrogenation, catalyzed by thermally stable Irn (n = 8, 12, 18) atomic clusters that exhibit a cuboid structure, was studied using the B3LYP method with triple-ζ basis sets. Relativistic effects and dispersion corrections were included in the calculations. In the dehydrogenation reaction Irn + C2H6 → H-Irn-C2H5 → (H)2-Irn-C2H4, the first H-elimination is the rate-limiting step, primarily because the reaction releases sufficient heat to facilitate the second H-elimination. The catalytic activity of the Ir clusters strongly depends on the Ir cluster size and the specific catalytic site. Cubic Ir8 is the least reactive toward H-elimination in ethane: Ir8 + C2H6 → H-Ir8-C2H5 has a large (65 kJ/mol) energy barrier, whereas Ir12 (3 × 2 × 2 cuboid) and Ir18 (3 × 3 × 2 cuboid) lower this energy barrier to 22 and 3 kJ/mol, respectively. The site dependence is as prominent as the size effect. For example, the energy barrier for the Ir18 + C2H6 → H-Ir18-C2H5 reaction is 3, 48, and 71 kJ/mol at the corner, edge, or face-center sites of the Ir18 cuboid, respectively. Energy release due to Ir cluster insertion into an ethane C-H bond facilitates hydrogen migration on the Ir cluster surface, and the second H-elimination of ethane. In an oxygen-rich environment, oxygen molecules may be absorbed on the Ir cluster surface. The oxygen atoms bonded to the Ir cluster surface may slightly increase the energy barrier for H-elimination in ethane. However, the adsorption of oxygen and its reaction with H atoms on the Ir cluster releases sufficient heat to yield an overall thermodynamically favored reaction: Irn + C2H6 + 1/2O2 → Irn + C2H4 + H2O. These results will be useful toward reducing the energy cost of ethane dehydrogenation in industry.

Schwann Cells Transplantation Improves Locomotor Recovery in Rat Models with Spinal Cord Injury: a Systematic Review and Meta-Analysis.

BACKGROUND/AIMS: Schwann cells (SCs) which were demonstrated to be responsible for axonal myelination and ensheathing are widely studied and commonly used for cell transplantation to treat spinal cord injury (SCI). We performed this meta-analysis to summarize the effects of SCs versus controls for locomotor recovery in rat models of traumatic SCI. METHODS: Studies of the BBB scores after transplantation of SCs were searched out from Pubmed, Cochrane Library Medline databases and analyzed by Review Manager 5.2.5. RESULTS: Thirteen randomized controlled animal trials were selected with 283 rats enrolled. The studies were divided to different subgroups by different models of SCI, different cell doses for transplantation, different sources of SCs and different transplantation ways. The pooled results of this meta-analysis suggested that SCs transplantation cannot significantly improve the locomotor recovery at a short time after intervention (1 week after transplantation) in both impacted and hemi-sected SCI models. However, at a longer time after intervention (3, 5-7 and over 8 weeks after transplantation), significant improvement of BBB score emerged in SCs groups compared with control groups. Subgroup analyses revealed that SCs transplantation can significantly promote locomotor recovery regardless of in high or low doses of cells, from different sources (isolated from sciatic nerves or differentiated from bone marrow stromal cells(BMSCs)) and with or without scaffolding. CONCLUSION: SCs seem to demonstrate substantial beneficial effects on locomotor recovery in a widely-used animal models of SCI.

Puerarin improves cardiac function through regulation of energy metabolism in Streptozotocin-Nicotinamide induced diabetic mice after myocardial infarction.

It is well recognized that the incidence of heart failure and the risk of death is high in diabetic patients after myocardial infarction (MI). Accumulating evidence showed that puerarin (PUE) has protecting function on both cardiovascular disease and diabetes. The aim of this study is to explore whether puerarin could improve cardiac function in diabetic mice after MI and the underlying mechanism. The left anterior of Streptozotocin (STZ)-Nicotinamide (NA) induced diabetic mice were ligated permanently except for the Shame group. Then the operated mice were randomly treated with PUE or saline. Cardiac function was evaluated by echocardiograph before and at 1, 2, 4 weeks after MI. GLUT4/CD36/p-Akt/PPAR α of the heart was examined after treatment for 4 weeks. The results indicated that PUE significantly increased survival rate, improved cardiac function compared with MI group. Moreover, PUE increased expression and translocation of GLUT4 while attenuated expression and translocation of CD36. Western blot analysis showed that PUE enhanced phosphorylation of Akt and decreased PPAR α. This study demonstrated that PUE improved cardiac function after MI in diabetic mice through regulation of energy metabolism, the possible mechanism responsible for the effect of PUE was increasing the expression and translocation of GLUT4 while attenuating the expression and translocation of CD36.

The diameters and number of nerve fibers in spinal nerve roots.

OBJECTIVE: To investigate the anatomical and histological features of spinal nerve roots and provide base data for neuroanastomosis therapy for paraplegia. METHODS: Spinal nerve roots from C1 to S5 were exposed on six adult cadavers. The diameter and the number of nerve fibers of each nerve root were measured, respectively, with a caliper and image analysis software. RESULTS: As for ventral roots, the diameter of C5 (2.50 ± 0.55 mm) was the largest in cervical segments. In thoracic and lumbosacral segments, the diameter gradually increased from T11 to S1 and then decreased from S1 to S5 except L3. S1 (1.43 ± 0.16 mm) was the thickest root and S5 (0.14 ± 0.02 mm) was the thinnest one. As for dorsal roots, the diameter of C7 (4.61 ± 0.87 mm) was the largest in cervical segments. From T11 to S1, the diameter increased and then decreased gradually from S1 to S5. The diameter of dorsal roots from T1 to S5 was largest at S1 (2.95 ± 0.57 mm) and smallest at S5 (0.27 ± 0.13 mm), respectively. C7 (8467 ± 1019), T12 (6538 ± 892), L3 (9169 ± 1160), and S1 (8253 ± 1419) ventral roots contained the most nerve fibers in cervical, thoracic, lumbar, and sacral segments, respectively. Similarly, C7 (39 653 ± 8458), T1 (26 507 ± 7617), L5 (34 455 ± 2740), and S1 (41 543 ± 3036) dorsal roots, respectively, contained the most nerve fibers in their corresponding segments. CONCLUSION: The findings in the current study provided the imperative data and may be valuable for spinal nerve root microanastomosis surgery in the paraplegic patients.

MicroRNA-200b stimulates tumour growth in TGFBR2-null colorectal cancers by negatively regulating p27/kip1.

Colorectal cancer (CRC) remains the most common malignancy worldwide. TGF-ß1 is often overexpressed in late stages of colorectal carcinogenesis and promotes tumour growth and metastasis. Several reports have verified that the loss of functional TGFBRII expression contributed to escape the tumour suppressor activity of TGF-ß1 and that the epithelial-to-mesenchymal transition (EMT) responded to TGF-ß1 involved in tumour invasion and metastasis. However, the mechanisms by which TGF-ß1 confers a growth advantage to TGFBRII-null colorectal cancer cells have not been elucidated. MicroRNAs (miRNAs) are post-transcriptional inhibitory regulators of gene expression that act by directly binding complementary mRNA and are key determinants of cancer initiation and progression. In this study, we revealed a role for miR-200b in colorectal cancer. MiR-200b was highly expressed in TGFBRII-null tumour tissues and colorectal cancer cell lines and positively correlated with cell proliferation in tumour tissues and cell lines. In contrast, decreasing the miR-200b level in TGFBRII-null cells suppressed cell proliferation and cell cycle progression. Furthermore, in vivo studies also suggested a stimulating effect of miR-200b on TGFBRII-null cell-derived xenografts. CDKN1B (p27/kip1) and RND3 (RhoE) have miR-200b binding sequences within their 3' untranslated regions and were confirmed to be direct targets of miR-200b using fluorescent reporter assays. Meanwhile, CDKN1B (p27/kip1) played a role in miR-200b-stimulated TGFBR-null CRC. This study suggests that miR-200b plays a tumour-promoting role by targeting CDKN1B (p27/kip1) in CRCs.

Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer.

BACKGROUND: Accumulating evidence indicates that the long non-coding RNA HOTAIR plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of HOTAIR in gastric carcinogenesis remains largely unknown. METHODS: HOTAIR expression was measured in 78 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of HOTAIR on gastric cancer cells were studied by overexpression and RNA interference approaches in vitro and in vivo. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation (RIP). The positive HOTAIR/HER2 interaction was identified and verified by immunohistochemistry assay and bivariate correlation analysis. RESULTS: HOTAIR upregulation was associated with larger tumor size, advanced pathological stage and extensive metastasis, and also correlated with shorter overall survival of gastric cancer patients. Furthermore, HOTAIR overexpression promoted the proliferation, migration and invasion of gastric carcinoma cells, while HOTAIR depletion inhibited both cell invasion and cell viability, and induced growth arrest in vitro and in vivo. In particular, HOTAIR may act as a ceRNA, effectively becoming a sink for miR-331-3p, thereby modulating the derepression of HER2 and imposing an additional level of post-transcriptional regulation. Finally, the positive HOTAIR/HER2 correlation was significantly associated with advanced gastric cancers. CONCLUSIONS: HOTAIR overexpression represents a biomarker of poor prognosis in gastric cancer, and may confer malignant phenotype to tumor cells. The ceRNA regulatory network involving HOTAIR and the positive interaction between HOTAIR and HER2 may contribute to a better understanding of gastric cancer pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.

MicroRNA-216b/Beclin 1 axis regulates autophagy and apoptosis in human Tenon's capsule fibroblasts upon hydroxycamptothecin exposure.

Proliferation and fibrosis of human Tenon's fibroblasts (HTFs) have significantly challenged the outcome of glaucoma filtration surgery. Hydroxycamptothecin (HCPT) is considered as a potential chemical to overcome this issue as it was previously shown that HCPT inhibited cell proliferation and induced apoptosis in fibroblasts. Here, we further dissected the molecular pathway, through which the HCPT inhibit the proliferation of HTFs. We showed that HCPT induced significant autophagy as well as apoptosis, two self-destructive processes, and down-regulated the expression of miR-216b in HTFs. Overexpression of miR-216b in HTFs suppressed the autophagy and apoptosis induced by HCPT, whereas silence of miR-216b led to effects that were similar to those caused by the treatment with HCPT. Further, we showed that miR-216b could directly target a specific fragment in the 3' untranslated region of Beclin 1 as demonstrated by luciferase assay, and consequently decreased the expression of Beclin 1. Consistently, knocking down Beclin 1 significantly decreased HCPT-triggered autophagy and apoptosis, and increased the viability of HTFs treated with HCPT, thus implicating that Beclin 1 functions as a pro-apoptotic molecule in this circumstance. Altogether, we concluded that miR-216b regulated both autophagy and apoptosis by modulating Beclin 1 in HTFs treated with HCPT. We also demonstrated that HCPT-induced autophagy is one of the agent's anti-proliferative effects.

PERK pathway are involved in NO-induced apoptosis in endothelial cells cocultured with RPE under high glucose conditions.

Although excessive nitric oxide (NO) induced from iNOS is critical for dysfunction of vascular endothelial cells (ECs) in the diabetic retina, its role on ECs injury remains unknown. RPE (retinal pigment epithelium) is the pigmented cell layer just outside the neurosensory retina that constitutes the blood-retinal-barrier (BRB) with ECs, and also serves as the limiting transport factor that maintains the retinal environment. Dysfunction of the RPE is related to oxidative stress that contributes to the progression of diabetic retina. Using a co-cultural biosystem, we demonstrate that NO generation and iNOS expression was increased in both ECs and RPE cells after high glucose treatment. Increased NO in ECs cocultured with RPE activate the endoplasmic reticulum (ER) and protein kinase RNA (PKR)-like ER kinase (PERK) pathway and involved in ECs apoptosis. Blockade of the iNOS pathway, or depletion of PERK effectively, reverses NO-mediated apoptosis. Our study demonstrates that iNOS and subsequently excessive NO generation in RPE cells can have an unanticipated effect by activating PERK pathways in ECs, resulting in a novel mechanism for vascular endothelium to avoid injury from prolonged hyperglycemia.

Pharmacological Inhibition of Gasdermin D Suppresses Angiotensin II-Induced Experimental Abdominal Aortic Aneurysms.

BACKGROUND: Gasdermin D, a molecule downstream of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing inflammasome, forms the membrane pore for the secretion of interleukin (IL)-1ß and IL-18, and also mediates pyroptosis. This study was to explore the influence of treatment with disulfiram, a small molecule inhibitor to gasdermin D, on the formation and progression of experimental abdominal aortic aneurysms (AAA). METHODS: AAAs were induced in 10-week-old male apolipoprotein E deficient mice by subcutaneous infusion of angiotensin II (1000 ng/min/kg body weight) for 28 days via osmotic minipumps. Three days prior to angiotensin II infusion, disulfiram (50 mg/kg) or an equal volume of saline as the vehicle control was administered daily via oral gavage. The influence on experimental AAAs was analyzed by serial measurements of aortic diameters via ultrasonography, grading AAA severity and histopathology at sacrifice. Serum IL-1ß and IL-18 levels, systolic blood pressure, total cholesterol, and triglyceride were also measured. Additional experiments assayed the influences on the cell viability and IL-1ß secretion of in vitro activated macrophages. RESULTS: Disulfiram significantly reduced the enlargement, incidence, and severity of angiotensin II-induced experimental AAAs with attenuation of medial elastin breaks, mural macrophage accumulation, and systolic blood pressure. The AAA suppression was also associated with reduced systemic levels of IL-1ß but not IL-18. However, disulfiram treatment had no impact on body weight gain and lipid levels in aneurysmal mice. Additionally, disulfiram treatment also markedly reduced the secretion of IL-1ß from activated macrophages with a limited effect on cell viability in vitro. CONCLUSIONS: Gasdermin D inhibition by disulfiram attenuated angiotensin II-induced experimental AAAs with reduced systemic IL-1ß levels and in vitro activated macrophage IL-1ß secretion. Our study suggests that pharmacological gasdermin D inhibition may have translational potential for limiting clinical AAA progression.

Treatment With Small Molecule Inhibitors of Advanced Glycation End-Products Formation and Advanced Glycation End-Products-Mediated Collagen Cross-Linking Promotes Experimental Aortic Aneurysm Progression in Diabetic Mice.

Background Although diabetes attenuates abdominal aortic aneurysms (AAAs), the mechanisms by which diabetes suppresses AAAs remain incompletely understood. Accumulation of advanced glycation end- (AGEs) reduces extracellular matrix (ECM) degradation in diabetes. Because ECM degradation is critical for AAA pathogenesis, we investigated whether AGEs mediate experimental AAA suppression in diabetes by blocking AGE formation or disrupting AGE-ECM cross-linking using small molecule inhibitors. Methods and Results Male C57BL/6J mice were treated with streptozotocin and intra-aortic elastase infusion to induce diabetes and experimental AAAs, respectively. Aminoguanidine (AGE formation inhibitor, 200 mg/kg), alagebrium (AGE-ECM cross-linking disrupter, 20 mg/kg), or vehicle was administered daily to mice from the last day following streptozotocin injection. AAAs were assessed via serial aortic diameter measurements, histopathology, and in vitro medial elastolysis assays. Treatment with aminoguanidine, not alagebrium, diminished AGEs in diabetic AAAs. Treatment with both inhibitors enhanced aortic enlargement in diabetic mice as compared with vehicle treatment. Neither enhanced AAA enlargement in nondiabetic mice. AAA enhancement in diabetic mice by aminoguanidine or alagebrium treatment promoted elastin degradation, smooth muscle cell depletion, mural macrophage accumulation, and neoangiogenesis without affecting matrix metalloproteinases, C-C motif chemokine ligand 2, or serum glucose concentration. Additionally, treatment with both inhibitors reversed suppression of diabetic aortic medial elastolysis by porcine pancreatic elastase in vitro. Conclusions Inhibiting AGE formation or AGE-ECM cross-linking enhances experimental AAAs in diabetes. These findings support the hypothesis that AGEs attenuate experimental AAAs in diabetes. These findings underscore the potential translational value of enhanced ECM cross-linking as an inhibitory strategy for early AAA disease.