TAK1 blockade as a therapy for retinal neovascularization.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-ß1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.

Approach for in vivo delivery of CRISPR/Cas system: a recent update and future prospect.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.

A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update.

Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.

Titanium dioxide nanoparticles impair the inner blood-retinal barrier and retinal electrophysiology through rapid ADAM17 activation and claudin-5 degradation.

BACKGROUND: Depending on their distinct properties, titanium dioxide nanoparticles (TiO2-NPs) are manufactured extensively and widely present in our daily necessities, with growing environmental release and public concerns. In sunscreen formulations, supplementation of TiO2-NPs may reach up to 25% (w/w). Ocular contact with TiO2-NPs may occur accidentally in certain cases, allowing undesirable risks to human vision. This study aimed to understand the barrier integrity of retinal endothelial cells in response to TiO2-NP exposure. bEnd.3 cells and human retinal endothelial cells (HRECs) were exposed to TiO2-NP, followed by examination of their tight junction components and functions. RESULTS: TiO2-NP treatment apparently induced a broken structure of the junctional plaques, conferring decreased transendothelial electrical resistance, a permeable paracellular cleft, and improved cell migration in vitro. This might involve rapid activation of metalloproteinase, a disintegrin and metalloproteinase 17 (ADAM17), and ADAM17-mediated claudin-5 degradation. For the in vivo study, C57BL/6 mice were administered a single dose of TiO2-NP intravitreally and then subjected to a complete ophthalmology examination. Fluorescein leakage and reduced blood flow at the optical disc indicated a damaged inner blood-retinal barrier induced by TiO2-NPs. Inappreciable change in the thickness of retinal sublayers and alleviated electroretinography amplitude were observed in the TiO2-NP-treated eyes. CONCLUSIONS: Overall, our data demonstrate that TiO2-NP can damage endothelial cell function, thereby affecting retinal electrophysiology.

The HDAC/HSP90 Inhibitor G570 Attenuated Blue Light-Induced Cell Migration in RPE Cells and Neovascularization in Mice through Decreased VEGF Production.

Age-related macular degeneration (AMD) occurs due to an abnormality of retinal pigment epithelium (RPE) cells that leads to gradual degeneration of the macula. Currently, AMD drug pipelines are endowed with limited options, and anti-VEGF agents stand as the dominantly employed therapy. Despite the proven efficacy of such agents, the evidenced side effects associated with their use underscore the need to elucidate other mechanisms involved and identify additional molecular targets for the sake of therapy improvement. The previous literature provided us with a solid rationale to preliminarily explore the potential of selective HDAC6 and HSP90 inhibitors to treat wet AMD. Rather than furnishing single-target agents (either HDAC6 or HSP90 inhibitor), this study recruited scaffolds endowed with the ability to concomitantly modulate both targets (HDAC6 and HSP90) for exploration. This plan was anticipated to accomplish the important goal of extracting amplified benefits via dual inhibition (HDAC6/HSP90) in wet AMD. As a result, G570 (indoline-based hydroxamate), a dual selective HDAC6-HSP90 inhibitor exerting its effects at micromolar concentrations, was pinpointed in the present endeavor to attenuate blue light-induced cell migration and retinal neovascularization by inhibiting VEGF production. In addition to the identification of a potential chemical tool (G570), the outcome of this study validates the candidate HDAC6-HSP90 as a compelling target for the development of futuristic therapeutics for wet AMD.

Ergosta-7,9(11),22-trien-3ß-ol Alleviates Intracerebral Hemorrhage-Induced Brain Injury and BV-2 Microglial Activation.

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by an exacerbation of neuroinflammation and neuronal injury, for which few effective therapies are available at present. Inhibition of excessive neuroglial activation has been reported to alleviate ICH-related brain injuries. In the present study, the anti-ICH activity and microglial mechanism of ergosta-7,9(11),22-trien-3ß-ol (EK100), a bioactive ingredient from Asian medicinal herb Antrodia camphorate, were evaluated. Post-treatment of EK100 significantly attenuated neurobehavioral deficit and MRI-related brain lesion in the mice model of collagenase-induced ICH. Additionally, EK100 alleviated the inducible expression of cyclooxygenase (COX)-2 and the activity of matrix metalloproteinase (MMP)-9 in the ipsilateral brain regions. Consistently, it was shown that EK100 concentration-dependently inhibited the expression of COX-2 protein in Toll-like receptor (TLR)-4 activator lipopolysaccharide (LPS)-activated microglial BV-2 and primary microglial cells. Furthermore, the production of microglial prostaglandin E2 and reactive oxygen species were attenuated by EK100. EK100 also attenuated the induction of astrocytic MMP-9 activation. Among several signaling pathways, EK100 significantly and concentration-dependently inhibited activation of c-Jun N-terminal kinase (JNK) MAPK in LPS-activated microglial BV-2 cells. Consistently, ipsilateral JNK activation was markedly inhibited by post-ICH-treated EK100 in vivo. In conclusion, EK100 exerted the inhibitory actions on microglial JNK activation, and attenuated brain COX-2 expression, MMP-9 activation, and brain injuries in the mice ICH model. Thus, EK100 may be proposed and employed as a potential therapeutic agent for ICH.

Hirsutanol A Attenuates Lipopolysaccharide-Mediated Matrix Metalloproteinase 9 Expression and Cytokines Production and Improves Endotoxemia-Induced Acute Sickness Behavior and Acute Lung Injury.

Activated human monocytes/macrophages, which increase the levels of matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, are the essential mechanisms for the progression of sepsis. In the present study, we determined the functions and mechanisms of hirsutanolA (HA), which is isolated from the red alga-derived marine fungus Chondrostereum sp. NTOU4196, on the production of pro-inflammatory mediators produced from lipopolysaccharide (LPS)-treated THP-1 cells. Our results showed that HA suppressed LPS-triggered MMP-9-mediated gelatinolysis and expression of protein and mRNA in a concentration-dependent manner without effects on TIMP-1 activity. Also, HA significantly attenuated the levels of TNF-α, IL-6, and IL-1ß from LPS-treated THP-1 cells. Moreover, HA significantly inhibited LPS-mediated STAT3 (Tyr705) phosphorylation, IκBα degradation and ERK1/2 activation in THP-1 cells. In an LPS-induced endotoxemia mouse model, studies indicated that HA pretreatment improved endotoxemia-induced acute sickness behavior, including acute motor deficits and anxiety-like behavior. HA also attenuated LPS-induced phospho-STAT3 and pro-MMP-9 activity in the hippocampus. Notably, HA reduced pathologic lung injury features, including interstitial tissue edema, infiltration of inflammatory cells and alveolar collapse. Likewise, HA suppressed the induction of phospho-STAT3 and pro-MMP-9 in lung tissues. In conclusion, our results provide pharmacological evidence that HA could be a useful agent for treating inflammatory diseases, including sepsis.

HADC8 Inhibitor WK2-16 Therapeutically Targets Lipopolysaccharide-Induced Mouse Model of Neuroinflammation and Microglial Activation.

Glial activation and neuroinflammatory processes play important roles in the pathogenesis of brain abscess and neurodegenerative diseases. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which contribute to the exacerbation of neuronal cell death. The inhibition of glial activation has been shown to alleviate neurodegenerative conditions. The present study was to investigate the specific HDAC8 inhibitor WK2-16, especially its effects on the neuroinflammatory responses through glial inactivation. WK2-16 significantly reduced the gelatinolytic activity of MMP-9, and expression of COX-2/iNOS proteins in striatal lipopolysaccharide (LPS)-induced neuroinflammation in C57BL/6 mice. The treatment of WK2-16 markedly improved neurobehavioral deficits. Immunofluorescent staining revealed that WK2-16 reduced LPS-stimulated astrogliosis and microglial activation in situ. Consistently, cellular studies revealed that WK2-16 significantly suppressed LPS-induced mouse microglia BV-2 cell proliferation. WK2-16 was proven to concentration-dependently induce the levels of acetylated SMC3 in microglial BV-2 cells. It also reduced the expression of COX-2/iNOS proteins and TNF-α production in LPS-activated microglial BV-2 cells. The signaling studies demonstrated that WK2-16 markedly inhibited LPS-activated STAT-1/-3 and Akt activation, but not NF-κB or MAPK signaling. In summary, the HADC8 inhibitor WK2-16 exhibited neuroprotective effects through its anti-neuroinflammation and glial inactivation properties, especially in microglia in vitro and in vivo.

Haloperidol Abrogates Matrix Metalloproteinase-9 Expression by Inhibition of NF-κB Activation in Stimulated Human Monocytic Cells.

Much evidence has indicated that matrix metalloproteinases (MMPs) participate in the progression of neuroinflammatory disorders. The present study was undertaken to investigate the inhibitory effect and mechanism of the antipsychotic haloperidol on MMP activation in the stimulated THP-1 monocytic cells. Haloperidol exerted a strong inhibition on tumor necrosis factor- (TNF-) α-induced MMP-9 gelatinolysis of THP-1 cells. A concentration-dependent inhibitory effect of haloperidol was observed in TNF-α-induced protein and mRNA expression of MMP-9. On the other hand, haloperidol slightly affected cell viability and tissue inhibition of metalloproteinase-1 levels. It significantly inhibited the degradation of inhibitor-κB-α (IκBα) in activated cells. Moreover, it suppressed activated nuclear factor-κB (NF-κB) detected by a mobility shift assay, NF-κB reporter gene, and chromatin immunoprecipitation analyses. Consistent with NF-κB inhibition, haloperidol exerted a strong inhibition of lipopolysaccharide- (LPS-) induced MMP-9 gelatinolysis but not of transforming growth factor-ß1-induced MMP-2. In in vivo studies, administration of haloperidol significantly attenuated LPS-induced intracerebral MMP-9 activation of the brain homogenate and the in situ in C57BL/6 mice. In conclusion, the selective anti-MMP-9 activation of haloperidol could possibly involve the inhibition of the NF-κB signal pathway. Hence, it was found that haloperidol treatment may represent a bystander of anti-MMP actions for its conventional psychotherapy.

A novel HDAC8 inhibitor H7E exerts retinoprotective effects against glaucomatous injury via ameliorating aberrant Müller glia activation and oxidative stress.

Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.

Oxygen-Induced Retinopathy in the Rat: An Animal Model to Study the Proliferative Retinal Vascular Pathology.

Diabetic retinopathy (DR) is one of the leading causes of vision loss worldwide. There are numerous animal models available for developing new ocular therapeutics and drug screening and to investigate the pathological processes involved in DR. Among those animal models, the oxygen-induced retinopathy (OIR) model, though originally developed as a model for retinopathy of prematurity, has also been used to investigate angiogenesis in proliferative DR with the phenomenon of ischemic avascular zones and pre-retinal neovascularization it demonstrated. Briefly, neonatal rodents are exposed to hyperoxia to induce vaso-obliteration. Upon removal from hyperoxia, hypoxia develops in the retina that eventually results in neovascularization. The OIR model is mostly used in small rodents such as mice and rats. Here, we describe a detailed experimental protocol of rat OIR model and the subsequent assessment of abnormal vasculature. By illustrating the vasculoprotective and anti-angiogenic activities of the treatment, OIR model might advance to a new platform for investigating novel ocular therapeutic strategies for DR.

Retinal protection by fungal product theissenolactone B in a sodium iodate-induced AMD model through targeting retinal pigment epithelial matrix metalloproteinase-9 and microglia activity.

Age-related macular degeneration (AMD) is the leading cause of low vision and blindness for which there is currently no cure. Increased matrix metalloproteinase-9 (MMP-9) was found in AMD and potently contributes to its pathogenesis. Resident microglia also promote the processes of chronic neuroinflammation, accelerating the progression of AMD. The present study investigates the effects and mechanisms of the natural compound theissenolactone B (LB53), isolated from Theissenia cinerea, on the effects of RPE dysregulation and microglia hyperactivation and its retinal protective ability in a sodium iodate (NaIO3)-induced retinal degeneration model of AMD. The fungal component LB53 significantly reduces MMP-9 gelatinolysis in TNF-α-stimulated human RPE cells (ARPE-19). Similarly, LB53 abolishes MMP-9 protein and mRNA expression in ARPE-19 cells. Moreover, LB53 efficiently suppresses nitric oxide (NO) production, iNOS expression, and intracellular ROS levels in LPS-stimulated TLR 4-activated microglial BV-2 cells. According to signaling studies, LB53 specifically targets canonical NF-κB signaling in both ARPE-19 and BV-2 microglia. In an RPE-BV-2 interaction assay, LB53 ameliorates LPS-activated BV-2 conditioned medium-induced MMP-9 activation and expression in the RPE. In NaIO3-induced AMD mouse model, LB53 restores photoreceptor and bipolar cell dysfunction as assessed by electroretinography (ERG). Additionally, LB53 prevents retinal thinning, primarily the photoreceptor, and reduces retinal blood flow from NaIO3 damage evaluated by optic coherence tomography (OCT) and laser speckle flowgraphy (LSFG), respectively. Our results demonstrate that LB53 exerts neuroprotection in a mouse model of AMD, which can be attributed to its anti-retinal inflammatory effects by impeding RPE-mediated MMP-9 activation and anti-microglia.

[The effect of pitavastatin on blood glucose and its efficacy in diabetic patients with hypercholesterolemia].

OBJECTIVE: To evaluate the effect of pitavastatin on blood glucose in patients with hypercholesterolemia, and to investigate the efficacy of pitavastatin in diabetic patients combined with hypercholesterolemia. METHOD: This study was a 12-week, multi-center, open-label, without parallel-group comparison, phase IV clinical trail. RESULTS: Contrasting to baseline, the prevalences at week 4 and 12 post-treatment of abnormal fasting plasma glucose (FPG) and glycosylated hemoglobin A1c (HbA1c) (FPG: 14.2%vs 14.1% and 11.0%; HbA1c: 14.3% vs 15.1% and 16.1%) in the safety set subjects without diabetes mellitus (DM), as well as in those with DM but not taking glucose-lowering drugs (FPG: 7/7 vs 4/7 and 5/7; HbA1c: 5/5 vs 4/4 and 5/5) had no significant changes (all P values > 0.05). Contrasting to baseline, the levels of TC [(6.51 ± 0.94) mmol/L vs (5.12 ± 0.93) mmol/L and (4.54 ± 1.00) mmol/L], LDL-C [(4.11 ± 0.79) mmol/L vs (3.02 ± 0.81) mmol/L and (2.51 ± 0.70) mmol/L] and TG [2.10 (1.53, 2.54) mmol/L vs 1.62 (1.26, 2.00) mmol/L and 1.35 (1.10, 1.86) mmol/L]at week 4 and 12 post-treatment in the per protocol set 55 subjects with DM were significantly reduced (all P values < 0.05); 33.3% of subjects at high risk and 10.0% of subjects at very high risk had achieved a TC target value; 55.6% of subjects at high risk and 40.0% of subjects at very high risk had achieved a LDL-C target value. CONCLUSION: Pitavastatin has a safe effect on blood glucose and it could be used to treat diabetic patients combined with hypercholesterolemia in China.

[Safety and efficacy of pitavastatin in patients with hypercholesterolemia: a multicenter study].

OBJECTIVE: To evaluate the safety and efficacy of pitavastatin in patients with hypercholesterolemia in China under conditions of extensive usage. METHODS: This was a 12-week, multicenter, open-label, without parallel-group comparison, phase IV clinical trial. RESULTS: There were 427 subjects in the safety set. The adverse events mainly included vomiting, myalgia and the elevations of aspartate transaminase (AST), alanine transaminase (ALT) and creatine kinase (CK), etc. The incidence of drug-related adverse events was 4.22%. There were no significant differences between pre-exposure and post-exposure average levels of renal function indicators and blood routine examination item (all P > 0.05). None of them had a high AST/ALT value, i.e. > 3 times upper limits of normal (ULN), or had a high CK value, i.e. > 10 times ULN. There were 397 subjects in the per protocol set. At week 12 post-treatment, the blood levels of total cholesterol and low density lipoprotein cholesterol (LDL-C) in subjects without previous treatment decreased 24.6% and 31.0% respectively, that of high density lipoprotein cholesterol (HDL-C) in subjects with HDL-C < 1.04 mmol/L increased 60.1% while that of triglyceride (TG) in subjects with TG > 1.70 mmol/L decreased 22.5% (P < 0.05). And 207 (92.3%) subjects were at a low risk, 46 (76.1%) subjects at an intermediate risk, 134 (47.8%) subjects at a high risk and 10 (40.0%) of subjects at a very high risk had achieved a LDL-C target value; the LDL-C goal achievement rate after switching from previous medication to pitavastatin was significant higher than that of pre-switching. CONCLUSION: Pitavastatin demonstrates positive safety and efficacy. It may be used for the treatment of patients with hypercholesterolemia in China.

[Effect and risk factors of pitavastatin on high sensitivity C-reactive protein in patients with hypercholesterolemia: a multilevel models analysis].

OBJECTIVE: To evaluate the effect of pitavastatin on high sensitivity C-reactive protein (hsCRP) in patients with hypercholesterolemia, and determine risk factors for the effect. METHODS: This study was a 12-week, multicenter, open-label, without parallel-group comparison, phase IV clinical trail. RESULTS: There were 330 subjects in the per protocol set. Contrast to the baseline, the average levels of hsCRP in all of subjects and the group without a history of receiving previous statin medication at week 12 post-treatment decreased respectively 26.4% (1.20 mg/L vs 1.68 mg/L) and 27.5% (1.21 mg/L vs 1.97 mg/L, all P < 0.05). The results of multilevel models indicated that the average levels of hsCRP reduced with the passage of treatment time, the time-varying rate of per-visit was 0.97 mg/L (95% confidence interval 0.96 - 0.98). Controlled individual background covariates, the model predicted that pulse pressure and white blood cell count on the baseline had the significant positive effects on hsCRP (P < 0.01). CONCLUSIONS: Pitavastatin decreases hsCRP in patients with hypercholesterolemia. The main risk factors for the effect are pulse pressure and white blood cell count on the baseline.

[Again review of research design and statistical methods of Chinese Journal of Cardiology].

OBJECTIVE: To re-evaluate and compare the research design and the use of statistical methods in Chinese Journal of Cardiology. METHOD: Summary the research design and statistical methods in all of the original papers in Chinese Journal of Cardiology all over the year of 2011, and compared the result with the evaluation of 2008. RESULTS: (1) There is no difference in the distribution of the design of researches of between the two volumes. Compared with the early volume, the use of survival regression and non-parameter test are increased, while decreased in the proportion of articles with no statistical analysis. (2) The proportions of articles in the later volume are significant lower than the former, such as 6(4%) with flaws in designs, 5(3%) with flaws in the expressions, 9(5%) with the incomplete of analysis. (3) The rate of correction of variance analysis has been increased, so as the multi-group comparisons and the test of normality. The error rate of usage has been decreased form 17% to 25% without significance in statistics due to the ignorance of the test of homogeneity of variance. CONCLUSION: Many improvements showed in Chinese Journal of Cardiology such as the regulation of the design and statistics. The homogeneity of variance should be paid more attention in the further application.

Topical application of TAK1 inhibitor encapsulated by gelatin particle alleviates corneal neovascularization.

Rationale: Corneal neovascularization (CoNV) is a severe complication of various types of corneal diseases, that leads to permanent visual impairment. Current treatments for CoNV, such as steroids or anti-vascular endothelial growth factor agents, are argued over their therapeutic efficacy and adverse effects. Here, we demonstrate that transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) plays an important role in the pathogenesis of CoNV. Methods: Angiogenic activities were assessed in ex vivo and in vitro models subjected to TAK1 inhibition by 5Z-7-oxozeaenol, a selective inhibitor of TAK1. RNA-Seq was used to examine pathways that could be potentially affected by TAK1 inhibition. A gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol was developed as the eyedrop to treat CoNV in a rodent model. Results: We showed that 5Z-7-oxozeaenol reduced angiogenic processes through impeding cell proliferation. Transcriptome analysis suggested 5Z-7-oxozeaenol principally suppresses cell cycle and DNA replication, thereby restraining cell proliferation. In addition, inhibition of TAK1 by 5Z-7-oxozeaenol blocked TNFα-mediated NFκB signalling, and its downstream genes related to angiogenesis and inflammation. 5Z-7-oxozeaenol also ameliorated pro-angiogenic activity, including endothelial migration and tube formation. Furthermore, topical administration of the gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol led to significantly greater suppression of CoNV in a mouse model compared to the free form of 5Z-7-oxozeaenol, likely due to extended retention of 5Z-7-oxozeaenol in the cornea. Conclusion: Our study shows the potential of TAK1 as a therapeutic target for pathological angiogenesis, and the gelatin nanoparticle coupled with 5Z-7-oxozeaenol as a promising new eyedrop administration model in treatment of CoNV.

An Integrative Multi-Omics Analysis Reveals MicroRNA-143 as a Potential Therapeutic to Attenuate Retinal Angiogenesis.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

NORAD promotes multiple myeloma cell progression via BMP6/P-ERK1/2 axis.

Multiple myeloma (MM) is one of the most common tumors of the hematological system and remains incurable. Recent studies have shown that long noncoding RNA NORAD is a potential oncogene in a variety of tumors. However, the general biological role and clinical value of NORAD in MM remains unknown. In this study, we measured NORAD expression in bone marrow of 60 newly diagnosed MM, 30 post treatment MM and 17 healthy donors by real-time quantitative polymerase chain reaction (qPCR). The NORAD gene was knockdown by lentiviral transfection in MM cell lines, and the effects of NORAD on apoptosis, cell cycle and cell proliferation in MM cells were examined by flow cytometry, CCK8 assay, EDU assay and Western blot, and the differential genes after knockdown of NORAD were screened by mRNA sequencing, followed by in vivo experiments and immunohistochemical assays. We found that knockdown of NORAD promoted MM cell apoptosis, induced cell cycle G1 phase arrest, and inhibited MM cell apoptosis in in vivo and in vitro experiments. Mechanistically, NORAD plays these roles through the BMP6/P-ERK1/2 axis. We discuss a novel mechanism by which NORAD acts pro-tumorigenically in MM via the BMP6/P-ERK1/2 axis.