Decreased Nitric Oxide Bioavailability Research Articles

Modulatory potentials of nitric oxide in obesity-induced endothelial dysfunction

Obesity is caused by a significant increase in adipose tissue due to excessive caloric intake that exceeds energy expenditure. Obesity has emerged as a significant public health issue in both poor resource and economic developed nations, owing to its increased incidence and links to chronic diseases, such osteoarthritis, hypertension, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases (CVDs). It is believed that uncoupled eNOS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, lipoxygenase, cyclooxygenase, microsomal P-450 enzymes, and pro-oxidant heme molecules are responsible for the drastic decrease in NO production and the relative increase in reactive oxygen species (ROS) secretion, which are considered as the molecular mechanisms associated with obesity-induced endothelial dysfunction. Vascular endothelium defines a monolayer of cells, which lies between the vascular smooth muscle cells (VSMCs) and the vessel lumen. It performs a number of protective actions facilitated by the release of nitric oxide (NO), a soluble gas that is generated in endothelial cells by the amino acid L-arginine through the action of endothelial nitric oxide synthase (eNOS). The maintenance of vascular homeostasis is facilitated by various biological actions of NO, such as modulation of vascular dilator tone, control of local cell growth, and protection of the endothelium from the harmful effects of cellular components in blood circulation, while maintaining normal endothelial functions. Decreased peripheral arterial endothelium-dependent dilation (EDD) in response to mechanical (intravascular shear) or chemical (acetylcholine) stimuli suggests that decreased NO bioavailability has been recognised as major molecular mechanisms associated with the progress and development of obesity-induced endothelial dysfunction. In this review, the modulatory effects of NO in obesity-induced endothelial dysfunction will be discussed.

Abstract P213: Mechanisms of Renin-Angiotensin System-Mediated Nitric Oxide Signaling in Podocytes

Introduction: The renin-angiotensin system (RAS) is a crucial cardiovascular and renal function regulator. Podocytes, glomerular epithelial cells that play a pivotal role in maintaining the renal filtration barrier, are key targets for RAS peptides. Our findings showed that RAS can promote calcium (Ca 2+ ) and nitric oxide (NO) signaling in podocytes. Moreover, a decrease in NO bioavailability directly correlates with glomerular filtration barrier damage, Ca 2+ overload, and the development of salt-sensitive hypertension. We hypothesized that RAS-mediated NO production is crucial for Ca 2+ control and cytoskeletal rearrangements in podocytes, contributing to glomerular filtration regulation. Methods: We performed live confocal imaging on human podocytes and freshly isolated rat glomeruli to detect the production of NO (DAF-FM) and Ca 2+ (Fluo-8). RAS peptides (Ang II, III, IV, 1-7, 1-9, and TRV120027) were used to stimulate cells, and NO and Ca 2+ production were measured. Blockers for Ang II Type 1 (AT1R), Ang II Type 2 (AT2R), and MAS receptors were used to identify the pathways involved in NO production. The dynamic changes in podocyte cytoskeleton mediated by RAS-NO intracellular signaling were detected using rhodamine-phalloidin staining and scanning ion microscopy. The NOS isoform-specific blockers were used to determine the source of NO in podocytes. ANOVA with post-hoc was used for comparisons. Results: Angiotensins II and III generate the most profound increases in NO levels in podocytes. Ang II, Ang III, and Ang 1-7 promote NO production by activating AT2R (n ≥ 20, p < 0.01). Interestingly, the increase in NO modulates AT1R- and β-arrestin-mediated Ca 2+ signaling (n ≥ 20, p < 0.05), suggesting the role of the NO/AT2R axis in Ca 2+ control in podocytes. NO production correlates with podocyte volume increase, and scanning ion microscopy experiments further confirmed that AT2R activation and corresponding NO elevation are responsible for the protrusion of podocyte's lamellipodia. Finally, we found that in podocytes, NOS1 is responsible for NO bioavailability, accounting for 69±5% of the total NO response, while the remaining NO levels are generated by NOS2 (39±5%). Conclusion: Our data show that RAS activation promotes NO production in podocytes, mainly via the AT2R pathway. NO is crucial for Ca 2+ control in podocytes and lamellipodia formation, suggesting the essential role of NO in glomerular filtration barrier function.

Hyperglycemia Induces Remodeling of Nitric Oxide Synthase Activity in Glomerular Podocytes

Background: The regulation of glomerular filtration is influenced by the renin-angiotensin system (RAS) and the release of nitric oxide (NO) by glomerular cells. Several studies suggest that NO levels in glomeruli decrease as renal pathology develops, and restoring NO levels can be beneficial for glomerular function. Despite these findings, the expression of nitric oxide synthase (NOS) subunits in podocytes, particularly under pathological conditions, still requires further investigation. We hypothesized that decreased NO bioavailability in diabetes and hyperglycemia is linked to pathological NOS remodeling, thus influencing redox balance in podocytes and promoting renal damage. Methods: Live-cell confocal imaging was used to investigate NO production and the distribution of NOS in both control and hyperglycemic conditions. First, we used immortalized human podocytes (obtained from M. Saleem lab) loaded with the DAF-FM fluorescent marker in control and hyperglycemic (exposed to high glucose for 12 hours) conditions. Next, freshly isolated glomeruli from Type 2 Diabetic Nephropathy (T2DN) and Wistar male rats (age 26-30 weeks) were used to confirm our data ex vivo. NO production was monitored following Angiotensin II (Ang II) stimulation, and the distribution of NOS was determined using specific NOS1 and NOS2 commercially available inhibitors (Nω-Propyl-L-arginine hydrochloride and L-NIL, respectively). One-way ANOVA (OriginPro) was used for statistical analysis. Results: Upon application of Ang II, rapid NO production was observed in cultured and Wistar rats isolated glomeruli podocytes. The release of NO was blocked by preincubation with corresponding inhibitors targeting NOS. Our findings reveal that NOS1 primarily contributes to signaling in healthy podocytes, accounting for 69±5% of the total NO response, while the remaining NO production mediated by the RAS is attributed to NOS2 (39±5%). In diabetic conditions, the maximal amplitude of NO production mediated by Ang II was decreased by 22±10% compared to the control (one-way ANOVA, n≥24, p<0.001). Furthermore, high glucose conditions significantly altered NOS distribution in podocytes, where NOS2 signaling became predominant (89±7% of the total NO response), and the contribution of NOS1 became less significant. Conclusions: The changes in the distribution of NO sources could be associated with heightened oxidative and nitrosative stress, promoting renal damage. Our research demonstrates that in diabetes, NOS2 activity becomes predominant, potentially playing a direct role in advancing pathological processes in podocytes. Revealing the mechanisms of this fundamental pathway in the glomerulus impacts our progress toward new treatment and preventive measures for diabetic kidney disease development. This research was supported by National Institutes of Health Grants R01 DK126720 (to OP), R01 DK129227 (to AS and OP), and R01 DK115749 (to KS), US Department of Veteran Affairs grants I01CX002391 (to JHL), I01CX001248 (JCO) and I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells.

Circulating uremic toxin indoxyl sulfate (IS), endothelial cell (EC) dysfunction, and decreased nitric oxide (NO) bioavailability are found in chronic kidney disease patients. NO nitrosylates/denitrosylates a specific protein's cysteine residue(s), forming S-nitrosothios (SNOs), and the decreased NO bioavailability could interfere with NO-mediated signaling events. We were interested in investigating the underlying mechanism(s) of the reduced NO and how it would regulate the S-nitrosylation of tissue transglutaminase (TG2) and its substrates on glycolytic, redox and inflammatory responses in normal and IS-induced EC injury. TG2, a therapeutic target for fibrosis, has a Ca2+-dependent transamidase (TGase) that is modulated by S-nitrosylation. We found IS increased oxidative stress, reduced NADPH and GSH levels, and uncoupled eNOS to generate NO. Immunoblot analysis demonstrated the upregulation of an angiotensin-converting enzyme (ACE) and significant downregulation of the beneficial ACE2 isoform that could contribute to oxidative stress in IS-induced injury. An in situ TGase assay demonstrated IS-activated TG2/TGase aminylated eNOS, NFkB, IkBα, PKM2, G6PD, GAPDH, and fibronectin (FN), leading to caspases activation. Except for FN, TGase substrates were all differentially S-nitrosylated either with or without IS but were denitrosylated in the presence of a specific, irreversible TG2/TGase inhibitor ZDON, suggesting ZDON-bound TG2 was not effectively transnitrosylating to TG2/TGase substrates. The data suggest novel roles of TG2 in the aminylation of its substrates and could also potentially function as a Cys-to-Cys S-nitrosylase to exert NO's bioactivity to its substrates and modulate glycolysis, redox, and inflammation in normal and IS-induced EC injury.

Sex-Dependent Impairment of Endothelium-Dependent Relaxation in Aorta of Mice with Overexpression of Hyaluronan in Tunica Media.

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.

The effect of altered nitric oxide bioavailability on the interaction of obesity and Alzheimer’s disease. Exploration of whether a high fat diet produces early cognitive decline in an Alzheimer’s mouse model.

AbstractBackgroundAlzheimer’s disease (AD) can be predicted by obesity and vascular risk factors. Obesity and AD are associated with vascular dysfunction, resulting in reduced peripheral and cerebral blood flow. Obesity is associated with reduced nitric oxide (NO) bioavailability, which contributes to reduced blood flow, the accumulation of β‐amyloid (Aβ), and eventual cognitive decline in AD. In animal models of AD, obesity or high fat diet exacerbates cognitive decline. The APPswe/PS1dE9 (APP/PS1) mouse, a model of beta amyloid neuropathology, demonstrates significant NO substrate dysfunction. NO substrate dysfunction and obesity potentially initiates metabolic and vascular dysfunction that can accelerate cognitive function decline. We investigated the impact of a high‐fat diet (HFD) on cognitive performance (discrimination reversal) in transgenic (Tg) and non‐transgenic (Non‐Tg) APP/PS1 mice prior to evaluating the role of NO in AD and obesity.MethodFemale APP/PS1 mice were split into four groups: Non‐Tg Chow (n = 9), Non‐Tg HFD (n = 5), Tg Chow (n = 9), and Tg HFD (n = 14). Mouse operant chambers with two nose poke ports and a dipper for delivering sucrose solution (15%) were used. Discrimination reversal procedure involved nose pokes in the left or right nose poke port produced access to 15% sucrose under a fixed ratio (FR) 1 schedule of reinforcement. Once the performance met criteria, the “correct” nose poke port was switched.ResultTg mice averaged fewer sessions to meet criteria in training. Non‐Tg and Tg mice on HFD failed to learn the discrimination to criteria and never experienced reversals. Non‐Tg mice on chow learned the discrimination multiple times. Only one Tg mice fed chow learned. Mice fed the chow diet received more sucrose reinforcers compared to mice fed the HFD.ConclusionAlthough HFD‐fed mice responded at lower rates, on average, they still made substantial numbers of responses suggesting that the failure to obtain the discrimination was not simply a result of a motivational deficit. Non‐Tg chow‐fed subjects made fewer errors than Tg chow‐fed subjects. Tg HFD‐fed mice originally made as many errors as their chow‐fed littermates but made more errors over the course of the study. Future research will investigate the impacts of decreased NO bioavailability.

Treatment with Resveratrol Reverses Priapism in the Sickle Cell Mouse

Introduction: Sickle cell disease (SCD) men display priapism. Townes and Berkeley SCD mice models are widely studied in basic sciences because they express 100% HbS in their circulating erythrocytes and exhibit features of priapism, and, as in SCD humans, this appears to be due to decreased nitric oxide (NO) signaling and increased oxidative stress, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, and penile erection persists (i.e., priapism) as cGMP is not degraded as a consequence of PDE5 downregulation. Moreover, oxidative/nitrosative stress has been associated with priapism in SCD mice.Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural phytoalexin product with potent antioxidant activity found in peanuts, berries, and grapes. In this study, since decreased NO bioavailability and increased oxidative stress leads to lower PDE5 in the penis, we aimed to evaluate the effects of resveratrol on functional and molecular alterations of erectile function in SCD mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress markers (4-HNE and 3-nitrotyrosine) in the erectile tissue of SCD. Methods: Wild type (WT, C57BL/6) and Townes SCD transgenic male mice (3-5 mo old) were treated with resveratrol (100 mg /kg/day) or its vehicle daily for two weeks via gavage. Corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation), were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (3-10 µM). Results: The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC50) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by resveratrol. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (4 Hz: 30 ± 8 and 80 ± 5 %, respectively; n=5), which was reversed by resveratrol treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (90 ± 1%) group compared to the WT mice (65 ± 6%), which were fully restored to WT values with resveratrol treatment (n=6). The mRNA expression for the PDE5 was reduced (P<0.05) by approximately 57% in penile tissues from SCD compared to the WT group, respectively. Resveratrol treatment restored (P<0.05) the mRNA expression of PDE5 in the penises from the SCD group (n=5-6). The protein expressions for 4-HNE and 3-nitrotyrosine (3-NT) were significantly higher (P<0.05) in erectile tissues from SCD compared to the WT group, which were reduced (P<0.05) by resveratrol treatment (n=6). In WT mice, PDE5, 4-HNE, and 3-NT protein expressions were not affected by resveratrol, as well as CC relaxations induced by ACh, SNP, and EFS. Conclusion: Resveratrol treatment reduced enhanced NO-mediated CC relaxations along with normalization of PDE5 expression and reduced oxidative stress markers expressions (3-NT and 4-HNE) in the penises from SCD. Therefore, resveratrol may constitute an additional strategy to prevent priapism in SCD. Financial Support: FAPESP.

Involvement of shedding induced by ADAM17 on the nitric oxide pathway in hypertension.

A Disintegrin and Metalloprotease 17 (ADAM17), also called tumor necrosis factor-ɑ (TNF-ɑ) convertase (TACE), is a well-known protease involved in the sheddase of growth factors, chemokines and cytokines. ADAM17 is also enrolled in hypertension, especially by shedding of angiotensin converting enzyme type 2 (ACE2) leading to impairment of angiotensin 1–7 [Ang-(1–7)] production and injury in vasodilation, induction of renal damage and cardiac hypertrophy. Activation of Mas receptor (MasR) by binding of Ang-(1–7) induces an increase in the nitric oxide (NO) gaseous molecule, which is an essential factor of vascular homeostasis and blood pressure control. On the other hand, TNF-ɑ has demonstrated to stimulate a decrease in nitric oxide bioavailability, triggering a disrupt in endothelium-dependent vasorelaxation. In spite of the previous studies, little knowledge is available about the involvement of the metalloprotease 17 and the NO pathways. Here we will provide an overview of the role of ADAM17 and Its mechanisms implicated with the NO formation.

Nitric oxide, aging and aerobic exercise: Sedentary individuals to Master's athletes

Aging is associated with a decline in physiological function and exercise performance. These effects are mediated, at least in part, by an age-related decrease in the bioavailability of nitric oxide (NO), a ubiquitous gasotransmitter and regulator of myriad physiological processes. The decrease in NO bioavailability with aging is especially apparent in sedentary individuals, whereas older, physically active individuals maintain higher levels of NO with advancing age. Strategies which enhance NO bioavailability (including nutritional supplementation) have been proposed as a potential means of reducing the age-related decrease in physiological function and enhancing exercise performance and may be of interest to a range of older individuals including those taking part in competitive sport. In this brief review we discuss the effects of aging on physiological function and endurance exercise performance, and the potential role of changes in NO bioavailability in these processes. We also provide a summary of current evidence for dietary supplementation with substrates for NO production — including inorganic nitrate and nitrite, l-arginine and l-citrulline — for improving exercise capacity/performance in older adults. Additionally, we discuss the (limited) evidence on the effects of (poly)phenols and other dietary antioxidants on NO bioavailability in older individuals. Finally, we provide suggestions for future research.

Flavored and Nicotine-Containing E-Cigarettes Induce Impaired Angiogenesis and Diabetic Wound Healing via Increased Endothelial Oxidative Stress and Reduced NO Bioavailability.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Pulmonary Hypertension Induces Sex Differences in Control of Contracting Rat Skeletal Muscle Oxygen Pressures

Pulmonary hypertension (PH) is a disease characterized by pulmonary vascular remodeling, cardiac impairments, and skeletal muscle dysfunction. Males and females display different features of the disease, including a higher incidence in females, while males present with greater mortality. Exercise intolerance is a cardinal manifestation of PH, but any potential differences regarding skeletal muscle function during contractions between males and females have not been studied. This investigation tested the hypothesis that sexual dimorphism exists in the oxygen delivery (Q̇O2)‐to‐oxygen utilization (V̇O2) matching with PH as represented by oxygen partial pressures (PO2) at rest and following the onset of contractions. Considering that females display greater reliance upon nitric oxide (NO) for vascular function and PH decreases NO bioavailability, we hypothesized that PH would attenuate the skeletal muscle PO2 response to a greater extent in females than males.MethodsThirty‐three Sprague‐Dawley rats were randomly assigned to healthy (HC) or PH groups. PH rats (male: n=7; female: n=9) were administered monocrotaline (MCT; 50 mg/kg, i.p.) while HC rats (male: n=9; female: n=8) were given saline. Disease progression was monitored via echocardiography. Phosphorescence quenching determined the O2 partial pressure in the interstitial space (PO2is) in the spinotrapezius muscle at rest and during contractions.ResultsAll PH rats displayed criteria for disease including right ventricular (RV) hypertrophy, elevated RV systolic pressure, and pulmonary congestion compared to HC. Indicators of PH severity did not differ between males and females (P&gt;0.05). Females displayed a greater reduction in baseline PO2is in PH versus HC when compared to male counterparts (‐29 ± 3% vs. ‐18 ± 2%; P&lt;0.05). No sex differences were present in PO2is kinetics (i.e., tau, amplitude) throughout contractions. However, females with PH reached a lower nadir in PO2is than PH males (4.2 ± 0.3 vs. 7.4 ± 0.4 mmHg; P&lt;0.05).ConclusionsThese findings support that Q̇O2‐to‐V̇O2 matching is impaired in a sex specific manner in skeletal muscle. Individual contributions of O2 delivery and utilization, as well as biochemical analysis of NO in skeletal muscle will be invaluable to elucidating the mechanistic bases for the effects seen herein.

NLRP3 Activation and Its Relationship to Endothelial Dysfunction and Oxidative Stress: Implications for Preeclampsia and Pharmacological Interventions.

Preeclampsia (PE) is a specific syndrome of human pregnancy, being one of the main causes of maternal death. Persistent inflammation in the endothelium stimulates the secretion of several inflammatory mediators, activating different signaling patterns. One of these mechanisms is related to NLRP3 activation, initiated by high levels of danger signals such as cholesterol, urate, and glucose, producing IL-1, IL-18, and cell death by pyroptosis. Furthermore, reactive oxygen species (ROS), act as an intermediate to activate NLRP3, contributing to subsequent inflammatory cascades and cell damage. Moreover, increased production of ROS may elevate nitric oxide (NO) catabolism and consequently decrease NO bioavailability. NO has many roles in immune responses, including the regulation of signaling cascades. At the site of inflammation, vascular endothelium is crucial in the regulation of systemic inflammation with important implications for homeostasis. In this review, we present the important role of NLRP3 activation in exacerbating oxidative stress and endothelial dysfunction. Considering that the causes related to these processes and inflammation in PE remain a challenge for clinical practice, the use of drugs related to inhibition of the NLRP3 may be a good option for future solutions for this disease.

Decreased nitric oxide content mediated by asymmetrical dimethylarginine and protein l-arginine methyltransferase 3 in macrophages induces trophoblast apoptosis: a potential cause of recurrent miscarriage.

Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal-foetal interface. Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. N/A.

Intravital assessment of precapillary pulmonary arterioles of type 1 diabetic mice shows oxidative damage and increased tone in response to NOS inhibition.

Microvascular dilation, important for peripheral tissue glucose distribution, also modulates alveolar perfusion and is inhibited by loss of bioavailable nitric oxide (NO) in diabetes mellitus (DM). We hypothesized that DM-induced oxidative stress decreases bioavailable NO and pulmonary precapillary arteriolar diameter, causing endothelial injury. We examined subpleural pulmonary arterioles after acute NO synthase (NOS) inhibition with NG-nitro-l-arginine methyl ester (l-NAME) in streptozotocin (STZ)- and saline (CTRL)-treated C57BL/6J mice. Microvascular changes were assessed by intravital microscopy in the right lung of anesthetized mice with open chest and ventilated lungs. Arteriolar tone in pulmonary arterioles (27.2-48.7 µm diameter) increased in CTRL mice (18.0 ± 11% constriction, P = 0.034, n = 5) but decreased in STZ mice (13.6 ± 7.5% dilation, P = 0.009, n = 5) after l-NAME. Lung tissue dihydroethidium (DHE) fluorescence (superoxide), inducible NOS expression, and protein nitrosylation (3-nitrotyrosine) increased in STZ mice and correlated with increased glucose levels (103.8 ± 8.8 mg/dL). Fluorescently labeled fibrinogen administration and fibrinogen immunostaining showed fibrinogen adhesion, indicating endothelial injury in STZ mice. In CTRL mice, vasoconstriction to l-NAME was likely due to the loss of bioavailable NO. Vasodilation in STZ mice may be due to decreased formation of a vasoconstrictor or emergence of a vasodilator. These findings provide novel evidence that DM targets the pulmonary microcirculation and that decreased NO bioavailability and increased precapillary arteriolar tone could potentially lead to ventilation-perfusion abnormalities, exacerbating systemic DM complications.NEW & NOTEWORTHY Diabetes pulmonary and microvascular consequences are well recognized but have not been characterized. We assessed lung microvascular changes in a live anesthetized mouse model of type 1 diabetes, using a novel intravital microscopy technique. Our results show new evidence that a diabetes-induced decrease in lung nitric oxide bioavailability underlies oxidative damage, enhanced platelet activation, and endothelial injury causing pulmonary microvascular dysfunction and altered vasoreactivity. These findings could provide novel strategies to prevent or reverse diabetes systemic consequences.

FXYD1 Is Protective Against Vascular Dysfunction.

[Figure: see text].

Abstract P086: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, And Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT), which is driven by a combination of renal tubular and vascular mechanisms. We have previously shown that disruption of Cullin3 (CUL3) in vascular smooth muscle impairs nitric oxide (NO) signaling and vasodilation through decreased cGMP bioavailability, strongly supporting a role of vascular CUL3 in blood pressure (BP) regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. Four weeks after tamoxifen, the resultant mice (E-Cul3Δ9) developed nocturnal HT (Night time peak systolic BP, E-Cul3Δ9: 135±3 vs Control: 124±3 mmHg) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s). No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting there was no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 ) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%) and cerebral resistance basilar artery (41% vs 77%). No difference in smooth muscle function was observed. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS, and NO production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). Thus, CUL3 mutations in the endothelium contribute to human HT in part through decreased NO bioavailability, endothelial dysfunction and secondary sympathoexcitation.

Abstract MP19: Smooth Muscle Cell Cytochrome B5 Reductase 3 Causes Heart Failure With Reduced Ejection Fraction Under Hypoxic Stress

Pulmonary hypertension causes increased pulmonary vascular resistance and right heart failure. Nitric oxide (NO) binds to its receptor soluble guanylyl cyclase (sGC) within vascular smooth muscle cells in its reduced heme (Fe 2+ ) form to increase intracellular cGMP production, activate protein kinase G signaling, and induce vessel relaxation. In pulmonary hypertension, endothelial damage leading to decreased NO bioavailability combined with oxidation of the sGC heme (Fe 3+ ) in vascular smooth muscle cells rendering it NO-insensitive results in vasonstriction. Notably, we have previously shown that cytochrome b5 reductase 3 (CYB5R3) in vascular smooth muscle cells is an sGC reductase (Fe 3+ to Fe 2+ ) that maintains NO-dependent vasodilation in vascular disease. We therefore hypothesized that CYB5R3 confers protection in pulmonary hypertension. To test this, we subjected smooth muscle cell-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 21 days of continuous hypoxia (10% O 2 ) and assessed vascular and cardiac function. We found that SMC CYB5R3 KO led to enhanced cardiac hypertrophy when compared to wild-type (WT) controls (n=8/ group). Specifically, SMC CYB5R3 KO mice had a larger right ventricle per tibia size, left ventricle mass, and Fulton index compared to WT (n=8/ group). Moreover, SMC CYB5R3 KO mice had a significantly impaired ejection fraction and fractional shortening, and increased left ventricular posterior wall pressure (n=3-5/group). No differences in right heart function or overall cardiac fibrosis were observed between groups (n=3-5/group). With respect to vascular function, hypoxic pulmonary arteries from SMC CYB5R3 KO mice also had a blunted response to sodium nitroprusside induced NO-dependent vasodilation though no difference in sGC activator BAY 58-2667 induced NO-independent vasodilation was observed as compared to WT (n=8-11/ group). No differences in pulmonary arterial sGC levels or medial area were observed between groups (n=6-7). Combined, these data implicate that loss of SMC CYB5R3 exacerbates cardiomyocyte hypertrophy and reduces cardiac function independent of pulmonary pressure differences.

Elevated circulating amyloid concentrations in obesity and diabetes promote vascular dysfunction.

Diabetes, obesity, and Alzheimer’s disease (AD) are associated with vascular complications and impaired nitric oxide (NO) production. Furthermore, increased β-site amyloid precursor protein–cleaving (APP-cleaving) enzyme 1 (BACE1), APP, and β-amyloid (Aβ) are linked with vascular disease development and increased BACE1 and Aβ accompany hyperglycemia and hyperlipidemia. However, the causal relationship between obesity and diabetes, increased Aβ, and vascular dysfunction is unclear. We report that diet-induced obesity (DIO) in mice increased plasma and vascular Aβ42 that correlated with decreased NO bioavailability, endothelial dysfunction, and increased blood pressure. Genetic or pharmacological reduction of BACE1 activity and Aβ42 prevented and reversed, respectively, these outcomes. In contrast, expression of human mutant APP in mice or Aβ42 infusion into control diet–fed mice to mimic obese levels impaired NO production, vascular relaxation, and raised blood pressure. In humans, increased plasma Aβ42 correlated with diabetes and endothelial dysfunction. Mechanistically, higher Aβ42 reduced endothelial NO synthase (eNOS), cyclic GMP (cGMP), and protein kinase G (PKG) activity independently of diet, whereas endothelin-1 was increased by diet and Aβ42. Lowering Aβ42 reversed the DIO deficit in the eNOS/cGMP/PKG pathway and decreased endothelin-1. Our findings suggest that BACE1 inhibitors may have therapeutic value in the treatment of vascular disease associated with diabetes.

The commonly‐used anthracycline chemotherapy drug Doxorubicin impairs vascular endothelial function via stimulation of mitochondrial superoxide

Doxorubicin (DOXO), a commonly used anthracycline chemotherapeutic agent, increases the risk of cardiovascular (CV) related morbidity and mortality. A key mechanism by which DOXO increases CV disease risk is by inducing endothelial dysfunction (impaired endothelium‐dependent dilation [EDD]) but: 1) the underlying mechanisms are unknown; 2) there are no established treatments for preventing these side effects.PurposeWe tested the hypotheses that: 1) DOXO impairs EDD via excess mitochondrial (mito) superoxide‐induced reductions in nitric oxide (NO) bioavailability; 2) these effects can be prevented with a mito‐targeted antioxidant.MethodsC57BL6 male mice (4mo of age) received one injection of either sham (PBS; n=4) or DOXO (10mg/ml; n=8). After 4 weeks, we assessed endothelial function (ex vivo carotid artery EDD to increasing doses of acetylcholine [ACh]) and potential underlying mechanisms: NO bioavailability (ACh dose response in the presence of the NO‐synthase inhibitor, L‐NAME); aortic total and mito‐specific superoxide production (electron paramagnetic resonance spectroscopy); aortic cytosolic, extracellular and mito isoforms of superoxide dismutase (SOD) (immunoblot); and the roles of excess total and mito‐specific superoxide in regulating EDD (ACh dose response in the presence of the SOD mimetic, TEMPOL, and the mito‐targeted antioxidant, MitoQ, respectively).ResultsMechanisms of ActionDOXO reduced maximal EDD by 41% (95±3 vs. 54±3 %; P=0.003). This reduction was mediated by a decrease in NO bioavailability, as EDD was not different between groups following incubation with L‐NAME. Vascular smooth muscle sensitivity to NO (dilatory response to sodium nitroprusside) was unaltered. DOXO increased aortic total superoxide production by 38% (5.3±0.7 to 7.3±0.5 amplitude units; P=0.003), without compensatory upregulation in aortic cytosolic or extracellular SOD. Reduced EDD with DOXO was mediated by excess superoxide production, as indicated by restoration of maximal EDD with TEMPOL (97±2%). DOXO increased aortic mito superoxide production by 50% (1.0±0.1 to 1.5±0.1 amplitude units; P=0.01) and induced a 60% reduction in aortic mito SOD (1.0±0.2 vs. 0.4±0.1 absorbance units; P=0.01). Reduced EDD with DOXO was mediated by increased mito superoxide production, as indicated by restoration of maximal EDD with MitoQ (91±8%).PreventionNext, we supplemented mice with MitoQ (250μM in drinking water; n=5) for 4 weeks following DOXO and showed that MitoQ prevented the reduction in EDD (97±1%, P=0.002 vs. DOXO alone) by preserving NO bioavailability via reduced mito superoxide (no added improvement with MitoQ incubation).ConclusionThese results suggest DOXO‐induced vascular endothelial dysfunction is mediated by excess mito superoxide‐induced reduction in NO bioavailability. Our results also demonstrate that MitoQ supplementation prevented DOXO‐induced endothelial dysfunction by suppressing mito superoxide and preserving NO bioavailability. Because MitoQ is available and safe for human use, there may be translational potential for reducing CV‐related morbidity and mortality in patients that have received DOXO chemotherapy.Support or Funding InformationNIH T32 DK007135‐44 (Z.S.C.)

Genetic Reduction of DNA Damage Repair Protein ATM Kinase Attenuates Endothelium‐Dependent Dilation and NO Bioavailability via Enhanced Superoxide in Old but not Young Mice

Advanced age is associated with accumulated DNA damage and disruption of selective DNA damage repair pathways which together can accelerate the aging phenotype. In the vasculature, aging is characterized by impaired endothelium‐dependent dilation (EDD) and reduced nitric oxide (NO) bioavailability. Activation of ataxia telangiectasia mutated (ATM) kinase, a key mediator of the response to double‐stranded DNA (dsDNA) breaks, leads to upregulation of its effectors p53 and p21. Importantly, expression of these proteins is negatively correlated to EDD and has been demonstrated to be increased in the endothelium with aging. While these findings suggest that dsDNA breaks impair EDD, it is unknown if alterations in ATM per se modulate EDD. We hypothesized that genetic deletion of ATM would impair endothelial function in young mice (4–6 mo, n=7–9/group) leading to a vascular phenotype that phenocopies aging. In young mice, EDD in response to acetylcholine (Ach) in isolated mesenteric arteries was similar between young ATM +/+ and ATM −/− mice in the absence or presence of the NO synthase inhibitor, L‐NAME (both p&gt;0.05) (Fig 1). Endothelium‐independent dilation (EID) to sodium nitroprusside (SNP) was also not different between young ATM +/+ and ATM −/− mice (p&gt;0.05). Thus, contrary to our hypothesis, EDD and NO were preserved despite ATM deletion, suggesting that an insufficient accumulation of DNA damage may have occurred in young mice to impair endothelial function. We next hypothesized that an additional genomic stressor, such as aging, may be necessary to uncover the effects of ATM deficiency on endothelial function. However, ATM −/− mice die at ~6 mo due to malignant thymic lymphomas, therefore, to explore the effects of an age‐related accumulation of DNA damage on endothelial function, we assessed Ach‐mediated EDD in mesenteric arteries of aged (18–19 mo) ATM +/+ and ATM +/− mice (n=5–13). While aging alone (4–6 vs. 18–19 mo, ATM +/+) only tended to reduce EDD (p=0.1), EDD was significantly blunted in old ATM +/− compared to age‐matched ATM +/+ (p&lt;0.01) (Fig 2A). This impairment in EDD resulted from decreased NO bioavailability, evidenced by a ~35% reduction in NO‐dependent vasodilation in aged ATM +/− compared to ATM +/+ mice (p&lt;0.01) (Fig 2B). EID to SNP did not differ between groups (p&gt;0.05). To determine if increases in reactive oxygen species may underlie blunted EDD/NO in aged ATM +/− mice, EDD was assessed in the presence of the superoxide scavenger, TEMPOL. TEMPOL restored EDD in aged ATM +/− mice (p&lt;0.05) to levels not different from young mice (p&gt;0.05). Here, we provide novel evidence that although ATM deficiency alone is insufficient to impair EDD in young mice, in the face of a known genomic stressor like aging, ATM deficiency leads to impaired EDD and reduced NO via increased superoxide. These findings support the concept that accumulation of DNA damage and genomic instability with advanced age impairs endothelial function.Support or Funding InformationFunding Information: Funded in part by awards from the NIH, R01 AG048366, R01 AG050238, R01 AG060395, K99 AT010017, T32 5T32HL139451‐02, and by US Department of Veterans Affairs I01BX004492ATM kinase deletion does not impair endothelium dependent dilation (EDD) or nitric oxide (NO) bioavailability in mesenteric arteries of young mice.EDD was assessed in response to increasing concentrations of acetylcholine (Ach) in isolated mesenteric arteries from young (4–6 mo) mice lacking ATM (ATM −/−) or wildtype (ATM +/+) littermate controls. To determine the contribution of NO to dilation, Ach responses were repeated in the presence of the NO synthase inhibitor, L‐NAME. †, difference after L‐NAME. p&lt;0.05. Data are means ± SEM.Figure 1ATM kinase deficiency impairs endothelium dependent dilation (EDD) and nitric oxide (NO) bioavailability in mesenteric arteries of aged mice.(A) EDD was assessed in response to increasing concentrations of acetylcholine (Ach) in isolated mesenteric arteries from aged (18–19 mo) mice heterozygous for ATM (ATM +/−) and wildtype (ATM +/+) controls. Ach dose‐responses were repeated in the presence of the NO synthase inhibitor, L‐NAME. (B) NO‐dependent vasodilation, i.e. the difference in maximal dilation to Ach in the absence or presence of L‐NAME, was calculated. *, difference from ATM+/+. †, difference after L‐NAME. p&lt;0.05. Data are means ± SEM.Figure 2