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.

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.

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.

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.

Methods for in vitro CRISPR/CasRx-Mediated RNA Editing.

Specific changes in the genome have been accomplished by the revolutionary gene-editing tool known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The advent of programmable RNA editing CRISPR/Cas nucleases has made this gene-editing tool safer and more precise. Specifically, CasRx, a family member of the Cas13d family, has shown great therapeutic potential. Here, we describe the in vitro methods of utilizing this powerful RNA editing platform and determine the RNA editing efficiencies for CasRx with different forms of guide RNAs (also known as gRNA or sgRNA).

Kinetics for the Slip Motion of Ripple Dislocations on Gold-Coated Poly(dimethylsiloxane).

Understanding the evolution of the defects in surface patterns is of practical importance to improve the performance and structural durability of the pattern-based micro- and nanodevices. In this work, we investigate the effects of temperature, compressive strain, and relative direction of the compression to the prestretch on the slip motion of ripple dislocations formed on the surface of gold-coated poly(dimethylsiloxane) films. Applying compression in the direction parallel to the direction of prestretch cannot cause the slip motion of the ripple dislocations. The initial velocity of the slip motion of the ripple dislocations increases with the increases in temperature and compressive strain. The temperature dependence of the ratios of the configuration force to the viscous coefficient and the viscous coefficient to the effective mass of the ripple dislocations follows the Arrhenius equation.

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.

Time-dependent deformation of artificial muscles based on Nylon 6.

Considering the potential applications of the Nylon 6 with thermal-induced deformation, we studied the creep deformation of non-twisted Nylon 6 wires and Nylon 6 artificial muscles as functions of annealing temperature. For comparison, we also studied the creep deformation of chicken muscle fibers in a temperature range of 20 to 35 °C. The experimental results showed that we could use the standard linear viscoelastic model to describe the creep deformation of the chicken muscle fibers, the non-twisted Nylon 6 wires, and the Nylon 6 artificial muscles. A simple method was developed to calculate the mechanical (elastic) constants and viscous resistance coefficient (viscosity) of the three different materials. The activation energy for the creep deformation of the chicken muscle fibers in the temperature of 20 to 35 °C was 18.79 kJ/mol. For the non-twisted Nylon 6 wires, the activation energy for the creep deformation was generally larger than that of the chicken muscle fibers, and was dependent on the annealing temperature. For the Nylon 6 artificial muscles, the activation energy for the creep deformation was smaller than that of the chicken muscle fibers.

WMJ-8-B, a novel hydroxamate derivative, induces MDA-MB-231 breast cancer cell death via the SHP-1-STAT3-survivin cascade.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors have been demonstrate to have broad-spectrum anti-tumour properties and have attracted lots of attention in the field of drug discovery. However, the underlying anti-tumour mechanisms of HDAC inhibitors remain incompletely understood. In this study, we aimed to characterize the underlying mechanisms through which the novel hydroxamate-based HDAC inhibitor, WMJ-8-B, induces the death of MDA-MB-231 breast cancer cells. EXPERIMENTAL APPROACH: Effects of WMJ-8-B on cell viability, cell cycle distribution, apoptosis and signalling molecules were analysed by the MTT assay, flowcytometric analysis, immunoblotting, reporter assay, chromatin immunoprecipitation analysis and use of siRNAs. A xenograft model was used to determine anti-tumour effects of WMJ-8-B in vivo. KEY RESULTS: WMJ-8-B induced survivin reduction, G2/M cell cycle arrest and apoptosis in MDA-MB-231 cells. STAT3 phosphorylation, transactivity and its binding to the survivin promoter region were reduced in WMJ-8-B-treated cells. WMJ-8-B activated the protein phosphatase SHP-1 and when SHP-1 signalling was blocked, the effects of WMJ-8-B on STAT3 phosphorylation and survivin levels were abolished. However, WMJ-8-B increased the transcription factor Sp1 binding to the p21 promoter region and enhanced p21 levels. Moreover, WMJ-8-B induced α-tubulin acetylation and disrupted microtubule assembly. Inhibition of HDACs was shown to contribute to WMJ-8-B's actions. Furthermore, WMJ-8-B suppressed the growth of MDA-MB-231 xenografts in mammary fat pads in vivo. CONCLUSIONS AND IMPLICATIONS: The SHP-1-STAT3-survivin and Sp1-p21 cascades are involved in WMJ-8-B-induced MDA-MB-231 breast cancer cell death. These results also indicate the potential of WMJ-8-B as a lead compound for treatment of breast cancer and warrant its clinical development.

Lovastatin causes FaDu hypopharyngeal carcinoma cell death via AMPK-p63-survivin signaling cascade.

Statins are used widely to lower serum cholesterol and the incidence of cardiovascular diseases. Growing evidence shows that statins also exhibit beneficial effects against cancers. In this study, we investigated the molecular mechanisms involved in lovastatin-induced cell death in Fadu hypopharyngeal carcinoma cells. Lovastatin caused cell cycle arrest and apoptosis in FaDu cells. Lovastatin increased p21(cip/Waf1) level while the survivin level was decreased in the presence of lovastatin. Survivin siRNA reduced cell viability and induced cell apoptosis in FaDu cells. Lovastatin induced phosphorylation of AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (MAPK) and transcription factor p63. Lovastatin also caused p63 acetylation and increased p63 binding to survivin promoter region in FaDu cells. AMPK-p38MAPK signaling blockade abrogated lovastatin-induced p63 phosphorylation. Lovastatin's enhancing effect on p63 acetylation was reduced in HDAC3- or HDAC4- transfected cells. Moreover, transfection of cells with AMPK dominant negative mutant (AMPK-DN), HDAC3, HDAC4 or p63 siRNA significantly reduced lovastatin's effects on p21(cip/Waf1) and survivin. Furthermore, lovastatin inhibited subcutaneous FaDu xenografts growth in vivo. Taken together, lovastatin may activate AMPK-p38MAPK-p63-survivin cascade to cause FaDu cell death. This study establishes, at least in part, the signaling cascade by which lovastatin induces hypopharyngeal carcinoma cell death.

Protein Phosphatase 2A in Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Murine Lymphatic Endothelial Cells.

The lymphatic endothelium plays an important role in the maintenance of tissue fluid homeostasis. It also participates in the pathogenesis of several inflammatory diseases. However, little is known about the underlying mechanisms by which lymphatic endothelial cell responds to inflammatory stimuli. In this study, we explored the mechanisms by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in murine lymphatic endothelial cells (SV-LECs). LPS caused increases in cox-2 mRNA and protein levels, as well as in COX-2 promoter luciferase activity in SV-LECs. These actions were associated with protein phosphatase 2A (PP2A), apoptosis signal-regulating kinase 1 (ASK1), JNK1/2 and p38MAPK activation, and NF-κB subunit p65 and C/EBPß phosphorylation. PP2A-ASK1 signaling blockade reduced LPS-induced JNK1/2, p38MAPK, p65 and C/EBPß phosphorylation. Transfection with PP2A siRNA reduced LPS's effects on p65 and C/EBPß binding to the COX-2 promoter region. Transfected with the NF-κB or C/EBPß site deletion of COX-2 reporter construct also abrogated LPS's enhancing effect on COX-2 promoter luciferase activity in SV-LECs. Taken together, the induction of COX-2 in SV-LECs exposed to LPS may involve PP2A-ASK1-JNK and/or p38MAPK-NF-κB and/or C/EBPß cascade.

Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells.

Inflammation and vascular perturbations are increasingly implicated in the pathogenesis of neurodegenerative diseases. Prevailing evidence suggests that valproic acid (VPA), an antiepileptic and mood stabilizer, exhibits not only neuro-protective effects, but also anti-inflammatory effects in neurodegenerative diseases. However, the underlying mechanism contributing to VPA's suppression of inflammatory responses remains unclear. In this study, we explored the inhibitory action of VPA on cyclooxygenase (COX)-2 expression in bEnd.3 mouse brain microvascular endothelial cells exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus. The LPS-induced increases in COX-2 protein level and COX-2 promoter-luciferase activity were significantly suppressed by VPA. VPA inhibited p38MAPK and JNK phosphorylation in LPS-stimulated bEnd.3 cells. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) or a JNK signaling inhibitor (JNK inhibitor II) significantly inhibited LPS-induced COX-2 expression. VPA inhibited LPS-induced NF-κB subunit p65 phosphorylation and κB-luciferase activity. LPS-increased p65 and C/EBPß binding to the COX-2 promoter region was attenuated in the presence of VPA. In addition, VPA suppression of p38MAPK, JNK and p65 phosphorylation, and subsequent COX-2 expression was restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. VPA also caused increases in MKP-1 acetylation and MKP-1 phosphatase activity in bEnd.3 cells. In conclusion, VPA may cause MKP-1 activation to dephosphorylate p38MAPK and JNK, leading to decrease in p65 and C/EBPß binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated bEnd.3 cells. The present study therefore supports the therapeutic value of VPA in alleviating brain inflammatory processes.