Effects of antisense oligonucleotide to iNOS on  hemodynamic and vascular changes induced by LPS

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries (≈250 μm) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion ofl-[3H]arginine tol-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.

Download Full-text

Differences in in vitro interactions between macrophages with pathogenic and environmental strains of Prototheca

Future Microbiology ◽

10.2217/fmb-2019-0238 ◽

2020 ◽

Vol 15 (6) ◽

pp. 427-436

Author(s):

Jin Yang ◽

Junhao Zhu ◽

Timothy Kudinha ◽

Fanrong Kong ◽

Qiang-qiang Zhang

Keyword(s):

Protein Expression ◽

Rt Pcr ◽

Inos Protein ◽

Colony Forming Unit ◽

Inos Protein Expression ◽

Cytokine Levels ◽

Different Strains ◽

Different Levels ◽

In Vitro Interactions

Aim: We investigated the interactions between macrophage and different strains of Prototheca. Materials & method: J774A.1 macrophages were infected with clinical isolates of Prototheca ciferrii 18125 and P. ciferrii 50779 and environmental isolate of P. ciferrii N71. Phagocytosis activities were compared by colony-forming unit assays at 3, 6 and 9 h after infection. Cytokine levels were detected by RT-PCR and ELISA. iNOS protein expression was examined by western blotting. Results: All P. ciferrii strains were phagocytized by macrophages but induced different levels of cytokines in macrophages. Moreover, infected by P. ciferrii N71 upregulated much higher iNOS protein expression in J774A.1 than that infected by the clinical strains. Conclusion: Clinical and environmental P. ciferrii strains show differences in their interactions with macrophages, which may be attributed to their virulence.

Download Full-text

iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation

Frontiers in Oncology ◽

10.3389/fonc.2021.631766 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhen Ding ◽

Dai Ogata ◽

Jason Roszik ◽

Yong Qin ◽

Sun-Hee Kim ◽

...

Keyword(s):

Nitric Oxide ◽

Protein Expression ◽

Nitrosative Stress ◽

Akt Pathway ◽

Melanoma Tumor ◽

Inos Protein ◽

Akt Activation ◽

Inos Protein Expression ◽

Patient Prognosis

We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO–driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

Download Full-text

Abstract 5460: Promoting Effect of 188 Re Radiation on the L-arginine/nitric Oxide Pathway in the Balloon Injured Rat Abdominal Aortic Arteries

Circulation ◽

10.1161/circ.118.suppl_18.s_554 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Guangzhen Zhong ◽

Xinchun Yang ◽

Pengrong Chen ◽

Chaoshu Tang

Keyword(s):

Protein Expression ◽

Vascular Lesions ◽

Inos Mrna ◽

Balloon Injury ◽

Inos Activity ◽

Inos Protein ◽

Abdominal Aortic ◽

Inos Protein Expression ◽

Aortic Artery ◽

Injured Artery

This study examined the effect of 188 Re radiation on L-Arginine(L-Arg)/NO pathway in the injured rat abdominal aortic artery and investigated the mechanism involved. Forty-three rats were randomly divided into 4 groups: control (n=10), 14 days after balloon injury without treatment (14B, n=10), 14 days after balloon injury plus radiation (14BR, n=13), radiation only (SR, n=10). Vascular lesions were created in the abdominal aortic arteries by overstretch balloon injury. Arteries were then left untreated or were treated with intra-aortic beta-radiation using 188 Re balloon to deliver 20Gy at 2 mm from the center of the source. Intima/medium(I/M) ratio of the artery were evaluated. Plasma NO, aortic NO generation, L-Arg transport, iNOS activity, iNOS mRNA and protein expression were studied. We found that I/M ratio in 14B group was higher than control (0.558±0.175 vs0.063±0.010, P<0.01), and higher than that in 14BR group (0.080±0.021, P<0.01). Plasma NO in each group was not significantly different with each other. NO generated by aortic artery in 14B group were 177% (P<0.01) higher than control (8.65±2.47μmol/mg protein) and 17% (P<0.05) lower than that in 14BR group. iNOS activity in the 14B group was 44% (P<0.01) higher than control(180±22 pmol.mg protein −1 .min −1 ) and 11%(P<0.05) lower than that in 14BR group. Quantitative RT-PCR showed that iNOS mRNA in 14B group was 290% (P<0.01) higher than control and 19%(P<0.05) lower than that in 14BR group. Immunohistochemistry results showed no iNOS protein expression in control. iNOS protein were similarly expressed in the 14B and 14BR groups (P>0.05). However iNOS protein was strongly expressed in the SR group. Incubated in the KH solution with L-Arg(100μmol/L), L-Arg transport efficacy in 14B group was 248% (P<0.01)higher than control(1.04±0.15pmol.g protein −1 .min −1 ), and 39%(P<0.01) higher than that in 14BR group. In summary, we found 188 Re inhibited neointima hyperplasia and promoted NO generation, iNOS activity and iNOS mRNA expression in the injured artery. However, it decreased L-Arg transport and had no significant influence on iNOS protein expression in the injured artery. Therefore, effect of radiation on the L-Arg/NO pathway explains partly the mechanism of intracoronary radiation.

Download Full-text

Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

Cell Transplantation ◽

10.1177/0963689720978739 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097873

Author(s):

Jing Li ◽

Youming Zhu ◽

Na Li ◽

Tao Wu ◽

Xianyu Zheng ◽

...

Keyword(s):

Protein Expression ◽

Dental Pulp ◽

Tube Formation ◽

Endothelial Differentiation ◽

Cell Source ◽

Lentiviral Infection ◽

Mrna And Protein Expression ◽

Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

Download Full-text

Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽

10.3390/v13020308 ◽

2021 ◽

Vol 13 (2) ◽

pp. 308

Author(s):

Ying-Ray Lee ◽

Chia-Ming Chang ◽

Yuan-Chieh Yeh ◽

Chi-Ying F. Huang ◽

Feng-Mao Lin ◽

...

Keyword(s):

Protein Expression ◽

Western Blotting ◽

Expression Profiles ◽

Plaque Assay ◽

Virus Titer ◽

Enterovirus 71 ◽

Luciferase Reporter ◽

Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

Download Full-text

708 Application of a novel mSENS drug delivery technology for mRNA therapeutics

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0708 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A750-A750

Author(s):

Sojin Lee ◽

Joon Young Park ◽

Goo-Young Kim ◽

Sang Woo Jo ◽

Minhyuk Yun ◽

...

Keyword(s):

Particle Size ◽

Protein Expression ◽

Cancer Vaccine ◽

Encapsulation Efficiency ◽

Tumor Model ◽

Cell Responses ◽

Delivery Platform ◽

Mrna Therapeutics

BackgroundSuccessful clinical translation of mRNA therapeutics requires an appropriate delivery strategy to overcome instability of mRNA and facilitate cellular uptake into the cells.1 Several lipid based nanoparticle approaches that encapsulate mRNA, notably lipid nanoparticle (LNP), have been developed, but their efficiency for delivery to certain target tissues and toxicity profiles still have room for improvement. The application of a novel polymer based nanoparticle technology platform, so called Stability Enhanced Nano Shells (SENS) for mRNA (mSENS) as a mRNA delivery platform for a cancer vaccine was demonstrated.MethodsThe physicochemical properties of mSENS formulation, particle size and encapsulation efficiency, were characterized using dynamic light scattering (DLS) and gel retardation assay. Using luciferase-encoding mRNA, the protein expression levels in vitro and in vivo were evaluated by luciferase assay or bioluminescence imaging (BLI), respectively. For cancer vaccine studies, antigen (tyrosinase-related protein 2 (Trp-2))-specific T cell responses were assessed by immunophenotyping mouse splenocytes using flow cytometry and by the enzyme-linked immunosorbent spot (ELISPOT) assay. The anti-tumor efficacy was studied in B16F10 lung tumor model in C57BL/6 mice. Liver and systemic toxicity of mSENS treated mice was evaluated through blood chemistry and complete blood count (CBC) tests.ResultsA library of mSENS formulations complexed with luciferase-encoding mRNA, were characterized for their particle size, surface charge, encapsulation efficiency, colloidal stability, and in vitro and in vivo luciferase protein expression level. Upon systemic administration in mice, varying biodistribution profiles were observed, implicating the potential for tailored delivery to target tissues. Particularly, cancer vaccine application was further developed leveraging the formulation with preferential spleen delivery. Following vaccination with Trp-2 mRNA encapsulated with mSENS (Trp-2 mRNA-mSENS) in B16F10 tumor bearing mice, strong Trp-2 antigen-specific IFN-γ T-cell responses were observed. Generated anti-tumor immunity also marked suppression of B16F10 lung tumors were observed in Trp-2-mSENS immunized mice compared to non-immunized controls, demonstrating the potential of mSENS as a mRNA delivery platform for the application for vaccine.ConclusionsProprietary biodegradable polymer based-mSENS platform offers an attractive delivery strategy for mRNA by tailoring to specific therapeutic applications. Depending on the application, whether it’s a vaccine or protein replacement, a rationally designed mSENS formulation can efficiently distribute mRNA to specific tissues. In particular, application of a splenic mSENS formulation for a cancer vaccine has been demonstrated in murine tumor model. In summary, mRNA delivery through mSENS platform is expected to provide significant opportunities in clinical development for mRNA therapeutics.Ethics ApprovalThe study was approved by Samyang Biopharmaceuticals’ IACUC (Institutional Animal Care and Use Committee), approval number SYAU-2027.ReferencePiotr S. Kowalski, Arnab Rudra, Lei Miao, and Daniel G. Anderson, delivering the messenger: advances in technologies for therapeutic mRNA delivery. Molecular Therapy Vol. 27 No 4 April 2019.

Download Full-text

Aloe-Emodin Relieves High-Fat Diet Induced QT Prolongation via MiR-1 Inhibition and IK1 Up-Regulation in Rats

Cellular Physiology and Biochemistry ◽

10.1159/000484120 ◽

2017 ◽

Vol 43 (5) ◽

pp. 1961-1973 ◽

Cited By ~ 11

Author(s):

Yan Bai ◽

Zhenli Su ◽

Hanqi Sun ◽

Wei Zhao ◽

Xue Chen ◽

...

Keyword(s):

Action Potential ◽

Protein Expression ◽

High Fat Diet ◽

Qt Prolongation ◽

Electrical Remodeling ◽

High Fat ◽

Treatment Groups ◽

Aloe Emodin

Background/Aims: High-fat diet (HFD) causes cardiac electrical remodeling and increases the risk of ventricular arrhythmias. Aloe-emodin (AE) is an anthraquinone component isolated from rhubarb and has a similar chemical structure with emodin. The protective effect of emodin against cardiac diseases has been reported in the literature. However, the cardioprotective property of AE is still unknown. The present study investigated the effect of AE on HFD-induced QT prolongation in rats. Methods: Adult male Wistar rats were randomly divided into three groups: control, HFD, and AE-treatment groups. Normal diet was given to rats in the control group, high-fat diet was given to rats in HFD and AE-treatment groups for a total of 10 weeks. First, HFD rats and AE-treatment rats were fed with high-fat diet for 4 weeks to establish the HFD model. Serum total cholesterol and triglyceride levels were measured to validate the HFD model. Afterward, AE-treatment rats were intragastrically administered with 100 mg/kg AE each day for 6 weeks. Electrocardiogram monitoring and whole-cell patch-clamp technique were applied to examine cardiac electrical activity, action potential and inward rectifier K+ current (IK1), respectively. Neonatal rat ventricular myocytes (NRVMs) were subjected to cholesterol and/or AE. Protein expression of Kir2.1 was detected by Western blot and miR-1 level was examined by real-time PCR in vivo and in vitro, respectively. Results: In vivo, AE significantly shortened the QT interval, action potential duration at 90% repolarization (APD90) and resting membrane potential (RMP), which were markedly elongated by HFD. AE increased IK1 current and Kir2.1 protein expression which were reduced in HFD rats. Furthermore, AE significantly inhibited pro-arrhythmic miR-1 in the hearts of HFD rats. In vitro, AE decreased miR-1 expression levels resulting in an increase of Kir2.1 protein levels in cholesterol-enriched NRVMs. Conclusions: AE prevents HFD-induced QT prolongation by repressing miR-1 and upregulating its target Kir2.1. These findings suggest a novel pharmacological role of AE in HFD-induced cardiac electrical remodeling.

Download Full-text

Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

Journal of Virology ◽

10.1128/jvi.79.24.15238-15245.2005 ◽

2005 ◽

Vol 79 (24) ◽

pp. 15238-15245 ◽

Cited By ~ 41

Author(s):

Alejandra E. Arbetman ◽

Michael Lochrie ◽

Shangzhen Zhou ◽

Jennifer Wellman ◽

Ciaran Scallan ◽

...

Keyword(s):

Protein Expression ◽

Neutralization Assay ◽

Biological Properties ◽

Scid Mice ◽

Factor Ix ◽

In Vivo Model ◽

Adeno Associated Virus ◽

Human Factor Ix

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

Download Full-text

Platelet-activating factor contributes to acute lung leak in rats given interleukin-1 intratracheally

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.1.l75 ◽

2000 ◽

Vol 279 (1) ◽

pp. L75-L80 ◽

Cited By ~ 29

Author(s):

Young M. Lee ◽

Brooks M. Hybertson ◽

Hyun G. Cho ◽

Lance S. Terada ◽

Okyong Cho ◽

...

Keyword(s):

Interleukin 1 ◽

Platelet Activating Factor ◽

Lavage Fluid ◽

Lung Lavage ◽

Myeloperoxidase Activity ◽

Cerium Chloride ◽

Neutrophil Accumulation ◽

Web 2086

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.

Download Full-text