DETA NONOate Research Articles

Anti-Quenching NIR-II Excitation Phenylboronic Acid Modified Conjugated Polyelectrolyte for Intracellular Peroxynitrite-Enhanced Chemo-Photothermal Therapy.

Multidrug resistance to clinical chemotherapeutic drugs severely limits antitumor efficacy and patient survival. The integration of chemotherapy with photothermal therapy (PTT) and reactive nitrogen species has become a major strategy to enhance cancer treatment efficacy. Herein, a multifunctional peroxynitrite (ONOO-) nanogenerator (PBT/NO/Pt) for NIR-II fluorescence (NIR-II FL)/NIR-II photoacoustic (NIR-II PA) imaging-guided chemo/NIR-II PTT/ONOO- combination therapy is reported. The multifunction nanogenerator is developed by co-loading a pH-sensitive nitric oxide donor (DETA NONOate) and nicotinamide adenine dinucleotide phosphate oxidases trigger superoxide (O2 •-) generator chemotherapy drug (CDDP) to an NIR-II excitation-conjugated polyelectrolyte (PNC11BA). PNC11BA has non-conjugated alkyl chain segments in the polymer backbone and abundant positively charged phenylboronic acid in its side chains, which support the anti-quenching of NIR-II FL and the integration of DETA NONOate and CDDP into PBT/NO/Pt. In the acidic tumor microenvironment, the coordination bonds between CDDP and PNC11BA are cleaved, releasing CDDP for chemotherapeutic activity. The simultaneous release of nitric oxide (NO) and O2 •- rapidly leads to the in situ generation of the more cytotoxic reactive physiological nitrogen species ONOO-. In vitro and in vivo results prove that PBT/NO/Pt exhibited a markedly ONOO- enhanced chemo-photothermal synergistic therapy for SKOV3/DDP tumor by downregulating the intracellular glutathione and increasing CDDP-DNA adducts.

Intratracheally Administered Peptide-Modified Lipid Admixture Containing Fasudil and/or DETA NONOate Ameliorates Various Pathologies of Pulmonary Arterial Hypertension.

This study examined the therapeutic potential of a combination therapy using fasudil, a Rho-kinase inhibitor, and DETA NONOate (DN), a nitric oxide donor, delivered as a lipid admixture modified with a cyclic homing peptide known as CAR (CAR-lipid mixture) for the treatment of pulmonary arterial hypertension (PAH). CAR-lipid mixtures were initially prepared via a thin-film hydration method and then combined with fasudil, DN, or a mixture of both. The therapeutic efficacy of this drug-laden lipid mixture was evaluated in a Sugen/Hypoxia (Su/Hx) rat model of PAH by measuring RV systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), Fulton indices, and assessing right ventricular (RV) functions, as well as evaluating pulmonary vascular morphology. Rats that received no treatment exhibited increases in RVSP, mPAP, Fulton indices, and changes in RV functional parameters. However, the treatment with the CAR-lipid mixture containing either fasudil or DN or a combination of both led to a decline in mPAP, RVSP, and Fulton indices compared to saline-treated rats. Similarly, rats that received these treatments showed concurrent improvement in various echocardiographic parameters such as pulmonary acceleration time (PAT), tricuspid annular plane systolic excursion (TAPSE), and ventricular free wall thickness (RVFWT). A significant decrease in the wall thickness of pulmonary arteries larger than 100 µm was observed with the combination therapy. The findings reveal that fasudil, DN, and their combination in a CAR-modified lipid mixture improved pulmonary hemodynamics, RV functions, and pathological alterations in the pulmonary vasculature. This study underscores the potential of combination therapy and targeted drug delivery in PAH treatment, laying the groundwork for future investigations into the optimization of these treatments, their long-term safety and efficacy, and the underlying mechanism of action of the proposed therapy.

Antimicrobial Hybrid Amphiphile via Dynamic Covalent Bonds Enables Bacterial Biofilm Dispersal and Bacteria Eradication

AbstractTo tackle the problems caused by bacterial biofilms, herein, this study reports an antimicrobial hybrid amphiphile (aHA) via dynamic covalent bonds for eradicating staphylococcal biofilms. aHA is synthesized via iminoboronate ester formation between DETA NONOate (nitric oxide donor), 3 4‐dihydroxybenaldehyde, and phenylboronic acid‐modified ciprofloxacin (Cip). aHA can self‐assemble in aqueous solution with an ultra‐small critical aggregation concentration of 3.80 × 10–5 mm and high drug loading content of 73.8%. The iminoboronate ester is sensitive to the acidic and oxidative biofilm microenvironment, liberating nitric oxide and Cip that can synergistically eradicate bacterial biofilms. To this end, aHA assemblies efficiently eradicate staphylococcal infections and ameliorate inflammation in the murine peritoneal and subcutaneous infection models without any notable side effects on normal tissues. Collectively, the aHA assemblies may provide a facile and efficient alternative to the current development of anti‐biofilm therapies.

A Comparative Study of the Impact of NO-Related Agents on MK-801- or Scopolamine-Induced Cognitive Impairments in the Morris Water Maze

Learning and memory deficits accompany numerous brain dysfunctions, including schizophrenia and Alzheimer's disease (AD), and many studies point to the role of nitric oxide (NO) in these processes. The present investigations constitute the follow-up of our previous research, in which we investigated the activity of NO releasers and a selective inhibitor of neuronal NO synthase (nNOS) to prevent short-term memory deficits in novel object recognition and T-maze. Here, the ability of the compounds to prevent the induction of long-term memory deficits by MK-801 or scopolamine administration was investigated. The Morris Water Maze test, a reliable and valid test of spatial learning and memory, was used, in which escape latency in the acquisition phase and nine different parameters in the retention phase were measured. A fast NO releaser (spermine NONOate), a slow NO releaser (DETA NONOate), and a nNOS inhibitor, N(ω)-propyl-L-arginine (NPLA), were used. The compounds were administered i.p. at a dose range of 0.05-0.5 mg/kg. All compounds prevented learning deficits in the acquisition phase and reversed reference memory deficits in the retention phase of the scopolamine-treated mice. Spermine NONOate was the least effective. In contrast, the drugs poorly antagonised MK-801-induced deficits, and only the administration of DETA NONOate induced some improvements in the retention trial.

Anti-Hypertensive Property of an NO Nanoparticle in an Adenine-Induced Chronic Kidney Disease Young Rat Model.

Hypertension is the most common complication of chronic kidney disease (CKD) in children but is still poorly controlled. Nitric oxide (NO) deficiency plays a pivotal role in CKD and hypertension. NO is known to have health benefits, while NO typically has a short half-life and is not specifically targeted. In this study, we used a pediatric CKD model, which was induced in young rats by feeding them 0.25% adenine. We investigated two different NO donors, namely S-nitrosoglutathione (GSNO) and diethylenetriamine/NO adduct (DETA NONOate) via intraperitoneal injection at 10 mg/kg/day daily for 3 weeks. GSNO was delivered by Cu2+-doped zeolitic imidazolate framework (Cu/ZIF-8) nanoparticles to generate NO. As a result, we observed Cu/ZIF-8 nanoparticles were successfully loaded with GSNO and were able to release NO. Young rats fed with adenine displayed kidney dysfunction and hypertension at 9 weeks of age, which were prevented by GSNO-loaded nanoparticle or DETA NONOate treatment. GSNO-loaded nanoparticles reduced CKD-induced hypertension, which was related to an enhanced endogenous NO-generating system, reduced renal oxidative stress, and downregulated several components belonging to the classic renin-angiotensin (RAS) system. Our results cast new light on targeting NO delivery through the use of nanoparticles aiming to improve child-focused outcomes related to CKD worthy of clinical translation.

Hypoxia-sensitive adjuvant loaded liposomes enhance the antimicrobial activity of azithromycin via phospholipase-triggered releasing for Pseudomonas aeruginosa biofilms eradication

Robust biofilms and the complex airway environment with thick sputum, local hypoxia and persistent inflammation induce the intractability of chronic pulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). Herein, we proposed a type of antibiotic-adjuvant liposomes (NANO@PS-LPs), co-incorporating azithromycin (AZI), adjuvant (2-nitroimidazole derivative, 6-NIH) and biofilm dispersant (nitric oxide donor, DETA NONOate). NANO@PS-LPs possessing negatively-charged surface and good hydrophilicity could easily penetrate through the sputum layer, then disassembled triggered by overexpressed phospholipase A2 (PLA2) in the microenvironment around biofilms. Nitric oxide produced by DETA NONOate promoted P. aeruginosa biofilms dispersal. 6-NIH was reduced to 2-aminomidazole derivative (6-AIH) under a hypoxic condition, and hence acted as an AZI adjuvant to enhance the antibacterial activity of AZI. It was found that NANO@PS-LPs could significantly eliminate mature P. aeruginosa biofilms, effectively kill dispersed bacteria, inhibit the metabolism of survivors and prevent P. aeruginosa adherence to airway epithelial cells, accordingly restrain recurrent infections. Additionally, NANO@PS-LPs performed a remarkable advantage in killing AZI-resistant P. aeruginosa and removing their biofilms. In summary, NANO@PS-LPs present a potential nano-strategy to treat stubborn pseudomonal pulmonary infections and overcome correlative drug resistance.

Abstract PD3-01: Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer

Abstract Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in TNBC patients. Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway is one of the most common oncogenic aberrations in TNBC. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). This study investigates the role of NO in PI3K-Akt pathway activation in human TNBC cells, in conditions with extracellular NO donor exposure and increased intracellular iNOS (NOS2) expression. TNBC cell lines BT549, HCC1937, Hs578T, SUM159, MDA-MB-231, and BT-20 were exposed to the NO donor DETA NONOate at increasing concentrations (0-500 µM) for 12 hours. Significant elevation in phosphorylation of Akt (Ser473 and Thr308) in response to increase NO donor concentration was found only in cell lines with wild-type PTEN and PIK3R1, such as SUM159, MDA-MB-231, and BT20. To evaluate whether NO-induced activation of Akt was PI3K-dependent, SUM159 (wild-type PTEN) and HCC1937 (PTEN mutant) cells were also pre-treated with PI3K inhibitor alpelisib before NO exposure. PI3K inhibition was only able to eliminate basal phospho-Akt and prevent NO-induced Akt activation in the SUM159 cell line. Furthermore, compared to vehicle control treated cells, we found that NO donor-induced activation enhanced phosphorylation in 15/18 Akt signaling proteins in SUM159 cells (PTEN intact) and 3/18 Akt signaling proteins in HCC1937 cells (PTEN loss). In Hs578T cells in which NOS2 was either overexpressed or knocked down via lentiviral transduction, we found that enhanced expression of NOS2 was associated with increased p-Akt expression and knock-down of NOS2 led to reduced p-Akt. This result suggests that iNOS, as opposed to other NOS isoforms, is more associated with modulating PI3K signaling in TNBC.We also analyzed TNBC data from The Cancer Genome Atlas and found that patients with positive iNOS mRNA expression had significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.0001] expression compared to patients with no iNOS expression. When we stratified this patient cohort based on both iNOS and PTEN expression, we only found significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.01] expression in patients with positive iNOS and PTEN expression, supporting the hypothesis that NO may impair PTEN’s function as a PI3K antagonist. Ongoing studies are directed to investigate mechanisms of iNOS-PTEN physical interactions, iNOS-dependent post-translational modifications to PTEN, and whether iNOS and PTEN are dual prognostic markers in TNBC patients. Citation Format: Tejaswini P. Reddy, Liliana Guzman-Rojas, Roberto R. Rosato, Wei Qian, Hong Zhao, Jenny C. Chang. Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-01.

Mechanism of Differential Susceptibility of Two (Canine Lung Adenocarcinoma) Cell Lines to 5-Aminolevulinic Acid-Mediated Photodynamic Therapy

Photodynamic therapy (PDT) is a clinically approved, minimally invasive treatment for malignant tumors. Protoporphyrin IX (PpIX), derived from 5-aminolevulinic acid (5-ALA) as the prodrug, is one of the photosensitizers used in PDT. Recently, we reported a significant difference in response to 5-ALA-mediated PDT treatment in two canine primary lung adenocarcinoma cell lines (sensitive to PDT: HDC cells, resistant to PDT: LuBi cells). This study aimed to examine the difference in cytotoxicity of 5-ALA-mediated PDT in these cells. Although intracellular PpIX levels before irradiation were similar between HDC and LuBi cells, the percentage of ROS-positive cells and apoptotic cells in LuBi cells treated with 5-ALA-mediated PDT was significantly lower than that in HDC cells treated with 5-ALA-mediated PDT. A high dosage of the NO donor, DETA NONOate, significantly increased the cytotoxicity of 5-ALA-mediated PDT against LuBi cells. These results suggest that the sensitivity of 5-ALA-mediated PDT might be correlated with NO.

Nitric Oxide-Dependent Electron Transport Chain Inhibition by the Cytochrome bc1 Inhibitor and Pretomanid Combination Kills Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc1 inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc1 inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.

Effect of RNF152 on NO induced apoptosis of colon cancer cells

Objective: To investigate the role and mechanism of ring finger protein 152 (RNF152) in the development of colitis-associated colon cancer (CAC). Methods: CAC was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Three different stages of mice during the development of colon cancer were obtained, named AD1, AD2 and AD3, respectively. A control group of mice without any treatment was set up as well. The expression of RNF152 in mouse colon tissues was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The effects of RNF152 overexpression on apoptosis and nitric oxide (NO) induced apoptosis was examined by flow cytometry. The expressions of Bcl-2 and Bcl-XL were detected by western blot. Results: CAC was effectively induced by AOM and DSS in C57BL/6 mice. The tumor incidence rate of AD3 group was 100%. The whole genome expression microarray data from mouse AOM-DSS model indicated that the mRNA level of RNF152 was gradually decreased during the development of colon cancer. The RT-qPCR results showed that RNF152 mRNA level in AD3 was 1.23±0.18, higher than 0.52±0.08 in negative control (P<0.01). Flow cytometry analysis showed that overexpression of RNF152 increased the apoptosis of RKO cells (P<0.01). The apoptotic rate of RKO-RNF152 cells treated with NO donor DETA NONOate was (31.2±3.1)%, higher than (14.2±2.1)% in RKO-PCDB cells (P<0.001). Overexpression of RNF152 significantly decreased the protein expressions of Bcl-XL and Bcl-2. Conclusion: Downregulation of RNF152 may facilitate the development of CAC by inhibiting the cell apoptosis.

Photoacoustic Cavitation‐Ignited Reactive Oxygen Species to Amplify Peroxynitrite Burst by Photosensitization‐Free Polymeric Nanocapsules

AbstractPhotoacoustic (PA) technology can transform light energy into acoustic wave, which can be used for either imaging or therapy that depends on the power density of pulsed laser. Here, we report photosensitizer‐free polymeric nanocapsules loaded with nitric oxide (NO) donors, namely NO‐NCPs, formulated from NIR light‐absorbable amphiphilic polymers and a NO‐releasing donor, DETA NONOate. Controlled NO release and nanocapsule dissociation are achieved in acidic lysosomes of cancer cells. More importantly, upon pulsed laser irradiation, the PA cavitation can excite water to generate significant reactive oxygen species (ROS) such as superoxide radical (O2.−), which further spontaneously reacts with the in situ released NO to burst highly cytotoxic peroxynitrite (ONOO−) in cancer cells. The resultant ONOO− generation greatly promotes mitochondrial damage and DNA fragmentation to initiate programmed cancer cell death. Apart from PA imaging, PA cavitation can intrinsically amplify reactive species via photosensitization‐free materials for promising disease theranostics.

Bifunctional polymer brush-grafted coronary stent for anticoagulation and endothelialization

At present, cardiovascular stent intervention faces clinical complications such as delayed endothelialization, late thrombosis and restenosis after implantation. In this work, a kind of bifunctional polymer brush-grafted coronary stent with anticoagulant and endothelial functions was developed. First, a block copolymer brush with zwitterionic structure consisting of sulfoethyl methacrylate (SBMA) and glycidyl methacrylate (GMA) was surface-induced grafted onto the surface of bare metal coronary stent by atom transfer radical polymerization. The diethylenetriamine NONOate (DETA NONOate), acted as nitric oxide (NO) donor to promote endothelialization, was then attached to polyglycidyl methacrylate (PGMA) brush by a reactive epoxy group to produce NO. The process of chemical modification and the release behavior of NO were characterized in detail. Moreover, the results of anticoagulant test, cytotoxicity test, endothelial cells (ECs) proliferation test and animal experiment of this bifunctional polymer brush-grafted coronary stent we proposed indicate that the zwitterion modified and NO supplied bifunctional coatings has good anticoagulant property, no cytotoxicity and significant endothelialization effect. This work opens the door to combine biological activity of NO and anticoagulant effect of zwitterions, which has great potential to address post-operative side effects associated with restenosis and late stent thrombosis.

Abstract B09: Nitric oxide stimulates PI3K-AKT pathway activation by S-nitrosylation of PTEN in human melanoma cells

Abstract Based on our previous finding that inflammation associated nitric oxide synthase (NOS) expression in melanoma and its association with poor prognosis in melanoma patients, we investigated the role of nitric oxide (NO) on the PI3K-Akt pathway. Human melanoma cell lines were exposed in vitro to the NO donor (DETA NONOate), and increased Akt Serine 473 phosphorylation (pAkt S473) was detected after addition of 50-500μM DETA NONOate at as early as 2 hours. Significant increases of pAkt S473 were observed mostly in cell lines with wild-type PTEN, such as A375 and MeWo, which under our conditions do not have elevated basal Akt phosphorylation. A375 cells were further examined for DETA NONOate-induced functional consequences of Akt phosphorylation and activation; enhanced kinase activity was found to increase phosphorylation of 12 of 16 AKT signaling proteins, which include GSK, MAPK, Bad, and others. By analyzing melanoma data in The Cancer Genome Atlas (TCGA), we found a similar result that patients with positive iNOS mRNA expression have higher pAkt S473 levels compared with those without iNOS. When both PTEN and iNOS expression status were analyzed, pAkt S473 is only significantly (P&amp;lt;0.05) increased in patients with positive iNOS and intact PTEN, supporting our hypothesis that NO may inhibit the role of PTEN as the PI3K antagonist. Further examination in A375 cells expressing iNOS confirmed the endogenous formation of PTEN S-nitrosylation (SNO), a NO-dependent post-translational modification. PTEN in cells treated with NO showed attenuated phosphatase activity. Consistent with this view, an analysis from stage III melanoma patient tissue microarray also revealed that patients with PTEN expression had a lower survival rate when they also had positive iNOS expression. In summary, our study revealed NO can stimulate the PI3K-Akt pathway in human melanoma cells, which could be promoted by NO-mediated PTEN SNO. These experimental discoveries are supported by clinical association data and have provided one plausible mechanistic insight into the possible functional consequences of inflammatory NO produced in human melanoma cells and microenvironment. Citation Format: Zhen Ding, Dai Ogata, Jason Roszik, Yong Qin, Sun-Hee Kim, Suhendan Ekmekcioglu, Elizabeth A. Grimm. Nitric oxide stimulates PI3K-AKT pathway activation by S-nitrosylation of PTEN in human melanoma cells [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B09.

DDAH1 Overexpression in Human Pulmonary Vascular Endothelial Cells Co‐Cultured with Human Pulmonary Artery Smooth Muscle Cells

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients as a result of pathologic vascular remodeling, and PH is an independent predictor of death in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the majority of ADMA in vivo and there are two isoforms, DDAH1 and DDAH2. Translational studies from our group analyzed a cohort of BPD patients and identified a potential biomarker, DDAH1 single nucleotide polymorphism (rs480414), that decreased the risk of PH in BPD by 61%. In the present study, we hypothesized that overexpression of DDAH1 in human pulmonary microvascular endothelial cells (hPMVEC) would lead to greater apoptosis and lower proliferation of human pulmonary artery smooth muscle cells (hPASMC) via effects in hPMVEC. We utilized a co‐culture system where hPMVEC were transfected with AdDDAH1 or AdGFP control for 24h and then incubated in co‐culture with hPASMC for 24h. Analysis included 1) western blot analysis for cleaved and total caspase‐3, ‐8, ‐9, p21, PCNA, and β‐actin and 2) trypan blue exclusion for viable cell numbers. Transfection of hPMVEC with AdDDAH1 resulted in greater DDAH1 protein expression (n=6, p=0.003) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found greater cleaved caspase‐3 (n=6, p=0.01), greater cleaved caspase‐8 (n=6, p=0.03), similar cleaved caspase‐9 (n=6, p=0.94), similar p21 (n=6, p=0.7), and similar PCNA (n=3, p=0.82) protein expression as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC, we found lower hPASMC viable cell numbers (n=3, p=0.008) as compared to control. After AdDDAH1 transfection of hPMVEC and co‐culture with hPASMC treated with DETA NONOate 250μM or DETA NONOate 250μM + Hemoglobin 100μM, the hPASMC viable cell numbers were lower compared to control (n=3, p=0.001; n=3, p=0.003, respectively). There was no difference detected between DETA NONOate 250μM and DETA NONOate 250μM + Hemoglobin 100μM groups (n=3, p=0.429). In conclusion, DDAH1 overexpression in hPMVEC resulted in greater apoptosis and lower proliferation in hPASMC, consistent with our hypothesis. We propose that DDAH1 is a potential new therapy for BPD‐PH via relieving pulmonary vascular endothelial ADMA‐inhibition of eNOS, resulting in greater endogenous NO production, greater pulmonary vascular wall apoptosis, and potentially less pathologic vascular remodeling. Future studies will determine if DDAH1 endothelial knockout mice have worse lung function as compared to wild‐type, after exposure to BPD‐PH model of LPS and hyperoxia.Support or Funding InformationK08 HL129080 (PI: Trittmann)

Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling.

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling.

Abstract P2039: Nitric Oxide Inhibition Enhances the Protein Expression of TNF-α Receptor Type 1 in Cultured M-1 Cells With Characteristics of Renal Collecting Duct Epithelium

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.

Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H2O2)-mediated oxidative stress

Non-steroidal anti-inflammatory drugs (NSAID) have shown promise as anticancer agents by inducing cell death apart from their antipyretic, anti-inflammatory and anti-thrombogenic effects. In our current study, we investigated the oxidative stress mediated cell death mechanism of a NSAID derivative NCX4040 (a nitric oxide (NO) releasing form of aspirin) in castration-resistant prostate cancer (CRPC) PC3 cell line. Our data revealed that NCX4040 is more potent than its parent compound aspirin or NO releasing compound DETA NONOate. NCX4040 significantly induced hydrogen peroxide formation with ensuing oxidative stress and mitochondrial depolarization resulting in lipid peroxidation, cell cycle arrest, inhibition of colony growth and induction of apoptosis in PC3 cells. Moreover, NCX4040 inhibited migration potential of PC3 cells by depolymerizing F-actin and promoting anoikis. Interestingly, elevated levels of NADPH oxidase 1 (NOX1), superoxide dismutase (SOD) 1 and 2 were observed upon NCX4040 treatment. However, down regulation of anti-apoptotic markers B-cell lymphoma 2 (Bcl2) and anti-oxidant thioredoxin reductase 1 (TXNRD1) expression were observed. In addition, NCX4040 down regulated cyclin D1 expression in PC3 cells further supporting the anticancer effect of NCX4040. Western blot analysis revealed that significant down regulation of key anti-apoptotic markers such as cellular inhibitor of apoptosis protein-1 (cIAP1), X-linked inhibitor of apoptosis (XIAP), survivin, and Cellular-Myc (c-Myc). On the other hand, NCX4040-treated cells showed upregulation of phosho histone H2AX (pH2AX), cleaved caspase3 and cleaved Poly [ADP-ribose] polymerase 1 (PARP1). Taken together, our data demonstrate that NCX4040 treatment enhances free radical formation which in turn induces oxidative stress leading to mitochondrial mediated cell death in metastatic PC3 cells.

Inhibition of NADP-malic enzyme activity by H2 S and NO in sweet pepper (Capsicum annuum L.) fruits.

NADPH is an essential cofactor in many physiological processes. Fruit ripening is caused by multiple biochemical pathways in which, reactive oxygen and nitrogen species (ROS/RNS) metabolism is involved. Previous studies have demonstrated the differential modulation of nitric oxide (NO) and hydrogen sulfide (H2 S) content during sweet pepper (Capsicum annuum L.) fruit ripening, both of which regulate NADP-isocitrate dehydrogenase activity. To gain a deeper understanding of the potential functions of other NADPH-generating components, we analyzed glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), which are involved in the oxidative phase of the pentose phosphate pathway (OxPPP) and NADP-malic enzyme (NADP-ME). During fruit ripening, G6PDH activity diminished by 38%, while 6PGDH and NADP-ME activity increased 1.5- and 2.6-fold, respectively. To better understand the potential regulation of these NADP-dehydrogenases by H2 S, we obtained a 50-75% ammonium-sulfate-enriched protein fraction containing these proteins. With the aid of in vitro assays, in the presence of H2 S, we observed that, while NADP-ME activity was inhibited by up to 29-32% using 2 and 5mM Na2 S as H2 S donor, G6PDH and 6PGDH activities were unaffected. On the other hand, NO donors, S-nitrosocyteine (CysNO) and DETA NONOate also inhibited NADP-ME activity by 35%. These findings suggest that both NADP-ME and 6PGDH play an important role in maintaining the supply of NADPH during pepper fruit ripening and that H2 S and NO partially modulate the NADPH-generating system.

Exogenous l-ARGININE does not stimulate production OF NO or cGMP within the rat corporal smooth muscle cells in culture

Background and aimNitric oxide (NO) is the intracellular chemical responsible for initiating a penile erection. Despite conflicting clinical data, it continues to be publicized and promoted that orally administered l-arginine, the putative substrate for NO, enhances the erectile response presumably by stimulating NO production by the corporal tissues resulting in an increase in cGMP production. To shed light on this issue, an in vitro study was conducted to explore the effect of direct exogenous administration of l-arginine as well as its precursor and metabolite, l-citrulline, on the NO-cGMP pathway within the cavernosal smooth muscle (CSM) cell. Materials and methodsCSM cells obtained from 8 to 10 week old Sprague-Dawley rats were grown in Dulbecco media with 20% fetal calf serum and then incubated with or without l-arginine (L-ARG) or l-citrulline (L-CIT) in a time course and dose-response manner. Sildenafil (0.4 mM), IBMX (1 mM), l-NAME (3 μM), ODQ (5 μM) and Deta Nonoate (10 μM) were used as either inhibitors or stimulators of the NO-cGMP pathway. mRNA and protein were extracted and used for the determination of the phosphodiesterase 5 (PDE5). PDE5 activity was determined by luminometry. cGMP content was determined by ELISA. Nitrite formation, an indicator of NO production, was measured in the cell culture media by a colorimetric assay. The cationic (CAT-1) and neutral (SNAT-1) amino acid transporters for L-ARG and L-CIT, respectively, were determined by Western blot. ResultsWhen compared to untreated CSM cells, incubation with 0.25–4.0 mM of L-ARG or 0.3–4.8 mM of L-CIT anywhere between 3 and 24 h did not result in any additional nitrite or cGMP production. The addition of l-NAME, IBMX or ODQ to these L-ARG and L-CIT treated cells did not alter these results. L-CIT but not L-ARG increased PDE5 mRNA and protein content as well as the activity of the PDE5 enzyme. Both CAT-1 and SNAT-1 were expressed in the CSM cells. ConclusionsThis in vitro study demonstrates that exogenous administration of L-ARG or L-CIT failed to stimulate production of either NO or cGMP by the corporal CSM cells. A re-evaluation of the presumptive role of the exogenous administration of L-ARG in improving the synthesis of NO at least at the level of the CSM cells appears warranted.

Cell Type‐Specific Differential Effects of Nitric Oxide on PDE3 Expression in Pulmonary Vasculature

Persistent pulmonary hypertension of the newborn (PPHN) is an important cause of morbidity and mortality in neonates. Often used in combination with hyperoxia, the pulmonary vasodilator, inhaled nitric oxide (iNO), is the only FDA‐approved therapy for PPHN, although up to 40% of patients are non‐responders. In the pulmonary vasculature, NO increases cGMP levels, promoting vasodilation. cGMP competitively inhibits phosphodiesterase 3 (PDE3), thereby decreasing cAMP hydrolysis and further enhancing vasodilation. However, in animal models, NO treatment has been shown to increase PDE3 expression and/or activity and has been postulated to be the reason for rebound PH upon iNO withdrawal. Furthermore, it has been reported that that addition of a PDE3 inhibitor to iNO enhances pulmonary vasorelaxation. AMP‐activated protein kinase (AMPK), a critical regulator of energy homeostasis with downstream effects on NO synthase (NOS), has been shown to be regulated by PDE3 isoforms in other cell types and implicated in PH pathogenesis. We hypothesize that NO regulates the AMPK pathway via modulating PDE3 activity in a cell‐type specific manner in the pulmonary vasculature. Human PASMC (hPASMC) were treated with the NO donor DETA NONOate (1 mM) or DMSO and incubated in 21% or 85% O2 for 48 h. PDE3A, PDE3B, phosphorylated (p) and total (T)‐AMPK, p‐ and T‐ACC, and β‐actin protein expression were quantified by Western blot analyses. A similar experiment was performed using hPMVEC. Additionally, p‐ and T‐endothelial NOS (eNOS) protein expression were quantified. Our results show that hPASMC PDE3A protein expression was increased following Deta NONOate treatment in normoxia and hyperoxia (5.5±0.4 ‐fold, p=0.0004; 7.5±0.8 ‐fold, p&lt;0.0001) relative to 21% O2 vehicle. Additionally, p/T‐AMPK protein expression was increased by 3‐fold after Deta NONOate treatment (p&lt;0.0001). Consistent with these results, p/T‐ACC (a downstream target of AMPK), was also increased with the addition of NO donor in both conditions. In hPMVEC, the addition of NO donor increased PDE3A protein expression (2.4±0.1 ‐fold, p&lt;0.0001). The combination of hyperoxia + NO donor, however, blunted the increase in PDE3A to a level similar to that in 21% O2, vehicle‐treated cells. Similarly, NO donor alone increased p/T‐AMPK (4.1±0.1 ‐fold, p&lt;0.0001), whereas the combination of hyperoxia + NO blunted this increase. Moreover, p‐eNOS/β‐actin and T‐eNOS/β‐actin were both increased with the NO donor (1.8±0.03 ‐fold, p&lt;0.0001; 2.9‐fold±0.20 SEM, p=0.0068), whereas the combination of hyperoxia + NO blunted the increase in p‐eNOS levels. In conclusion, exogenous NO donor increased PDE3A expression and AMPK activation in hPASMCs in both normoxia and hyperoxia. Alternatively, in hPMVEC, the increase in PDE3A expression, AMPK and eNOS activation after the addition of NO donor was blunted by hyperoxia. We speculate that these cell‐type specific differences in regulation of PDE3 and AMPK may be the reason for the variability in response of the pulmonary vasculature to PDE3 inhibition and AMPK modulators in PH.Support or Funding InformationNIH, NHLBI, R01, 1R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.