Plasma microrna quantification protocol.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

The Use of Acute Immunosuppressive Therapy to Improve Antibiotic Efficacy against Intracellular Staphylococcus aureus.

Interactions between Staphylococcus aureus and the host immune system can have significant impacts on antibiotic efficacy, suggesting that targeting and modulating the immune response to S. aureus infection may improve antibiotic efficacy and improve infection outcome. As we've previously shown, high levels of reactive oxygen species (ROS), associated with an M1-like proinflammatory macrophage response, potently induce antibiotic tolerance in S. aureus. Although the proinflammatory immune response is critical for initial control of pathogen burden, recent studies demonstrate that modulation of the macrophage response to an anti-inflammatory, or M2-like, response facilitates resolution of established S. aureus skin and soft tissue infections, arthritis, and bacteremia. Here, we evaluated the impact of host-directed immunosuppressive chemotherapeutics and anti-inflammatory agents on antibiotic efficacy against S. aureus. IMPORTANCE Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States with high rates of antibiotic treatment failure. Macrophages represent an important intracellular niche in experimental models of S. aureus bacteremia. Although a proinflammatory macrophage response is critical for controlling infection, previous studies have identified an antagonistic relationship between antibiotic treatment and the proinflammatory macrophage response. Reactive oxygen species, produced by macrophages during respiratory burst, coerce S. aureus into an antibiotic tolerant state, leading to poor treatment outcome. Here, we aimed to determine the potential of host-directed immunomodulators that reduce the production of reactive oxygen species to improve antibiotic efficacy against intracellular S. aureus.

Antioxidant Response Activating nanoParticles (ARAPas) localize to atherosclerotic plaque and locally activate the Nrf2 pathway.

Atherosclerotic disease is the leading cause of death world-wide with few novel therapies available despite the ongoing health burden. Redox dysfunction is a well-established driver of atherosclerotic progression; however, the clinical translation of redox-based therapies is lacking. One of the challenges facing redox-based therapies is their targeted delivery to cellular domains of redox dysregulation. In the current study, we sought to develop Antioxidant Response Activating nanoParticles (ARAPas), encapsulating redox-based interventions, that exploit macrophage biology and the dysfunctional endothelium in order to selectively accumulate in atherosclerotic plaque. We employed flash nanoprecipitation (FNP) to synthesize bio-compatible polymeric nanoparticles encapsulating the hydrophobic Nrf2 activator drug, CDDO-Methyl (CDDOMe-ARAPas). Nuclear factor erythroid 2-related factor 2 (Nrf2)-activators are a promising class of redox-active drug molecules whereby activation of Nrf2 results in the expression of several antioxidant and cyto-protective enzymes that can be athero-protective. In this study, we characterize the physicochemical properties of CDDOMe-ARAPas as well as confirm their in vitro internalization by murine macrophages. Drug release of CDDOMe was determined by Nrf2-driven GFP fluorescence. Moreover, we show that these CDDOMe-ARAPas exert anti-inflammatory effects in classically activated macrophages. Finally, we show that CDDOMe-ARAPas selectively accumulate in atherosclerotic plaque of two widely-used murine models of atherosclerosis: ApoE-/- and LDLr-/- mice, and are capable of increasing gene expression of Nrf2-transcriptional targets in the atherosclerotic aortic arch. Future work will assess the therapeutic efficacy of intra-plaque Nrf2 activation with CDDOMe-ARAPas to inhibit atherosclerotic plaque progression. Overall, our present studies underline that targeting of atherosclerotic plaque is an effective means to enhance delivery of redox-based interventions.

Polyamine-Conjugated Nitroxides Are Efficacious Inhibitors of Oxidative Reactions Catalyzed by Endothelial-Localized Myeloperoxidase.

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical (•NO2). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO's substrate hydrogen peroxide (H2O2) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on •NO2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Delivery of Cinnamic Aldehyde Antioxidant Response Activating nanoParticles (ARAPas) for Vascular Applications.

Selective delivery of nuclear factor erythroid 2-related factor 2 (Nrf2) activators to the injured vasculature at the time of vascular surgical intervention has the potential to attenuate oxidative stress and decrease vascular smooth muscle cell (VSMC) hyperproliferation and migration towards the inner vessel wall. To this end, we developed a nanoformulation of cinnamic aldehyde (CA), termed Antioxidant Response Activating nanoParticles (ARAPas), that can be readily loaded into macrophages ex vivo. The CA-ARAPas-macrophage system was used to study the effects of CA on VSMC in culture. CA was encapsulated into a pluronic micelle that was readily loaded into both murine and human macrophages. CA-ARAPas inhibits VSMC proliferation and migration, and activates Nrf2. Macrophage-mediated transfer of CA-ARAPas to VSMC is evident after 12 h, and Nrf2 activation is apparent after 24 h. This is the first report, to the best of our knowledge, of CA encapsulation in pluronic micelles for macrophage-mediated delivery studies. The results of this study highlight the feasibility of CA encapsulation and subsequent macrophage uptake for delivery of cargo into other pertinent cells, such as VSMC.

Light sheet fluorescence microscopy as a new method for unbiased three-dimensional analysis of vascular injury.

AIMS: Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. METHODS AND RESULTS: The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. CONCLUSION: LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

Myeloperoxidase: A versatile mediator of endothelial dysfunction and therapeutic target during cardiovascular disease.

Myeloperoxidase (MPO) is a prominent mammalian heme peroxidase and a fundamental component of the innate immune response against microbial pathogens. In recent times, MPO has received considerable attention as a key oxidative enzyme capable of impairing the bioactivity of nitric oxide (NO) and promoting endothelial dysfunction; a clinically relevant event that manifests throughout the development of inflammatory cardiovascular disease. Increasing evidence indicates that during cardiovascular disease, MPO is released intravascularly by activated leukocytes resulting in its transport and sequestration within the vascular endothelium. At this site, MPO catalyzes various oxidative reactions that are capable of promoting vascular inflammation and impairing NO bioactivity and endothelial function. In particular, MPO catalyzes the production of the potent oxidant hypochlorous acid (HOCl) and the catalytic consumption of NO via the enzyme's NO oxidase activity. An emerging paradigm is the ability of MPO to also influence endothelial function via non-catalytic, cytokine-like activities. In this review article we discuss the implications of our increasing knowledge of the versatility of MPO's actions as a mediator of cardiovascular disease and endothelial dysfunction for the development of new pharmacological agents capable of effectively combating MPO's pathogenic activities. More specifically, we will (i) discuss the various transport mechanisms by which MPO accumulates into the endothelium of inflamed or diseased arteries, (ii) detail the clinical and basic scientific evidence identifying MPO as a significant cause of endothelial dysfunction and cardiovascular disease, (iii) provide an up-to-date coverage on the different oxidative mechanisms by which MPO can impair endothelial function during cardiovascular disease including an evaluation of the contributions of MPO-catalyzed HOCl production and NO oxidation, and (iv) outline the novel non-enzymatic mechanisms of MPO and their potential contribution to endothelial dysfunction. Finally, we deliver a detailed appraisal of the different pharmacological strategies available for targeting the catalytic and non-catalytic modes-of-action of MPO in order to protect against endothelial dysfunction in cardiovascular disease.

Nanotherapies for Treatment of Cardiovascular Disease: A Case for Antioxidant Targeted Delivery.

PURPOSE OF REVIEW: Cardiovascular disease (CVD) involves a broad range of clinical manifestations resulting from a dysfunctional vascular system. Overproduction of reactive oxygen and nitrogen species are causally implicated in the severity of vascular dysfunction and CVD. Antioxidant therapy is an attractive avenue for treatment of CVD associated pathologies. Implementation of targeted nano-antioxidant therapies has the potential to overcome hurdles associated with systemic delivery of antioxidants. This review examines the currently available options for nanotherapeutic targeting CVD, and explores successful studies showcasing targeted nano-antioxidant therapy. RECENT FINDINGS: Active targeting strategies in the context of CVD heavily focus on immunotargeting to inflammatory markers like cell adhesion molecules, or to exposed extracellular matrix components. Targeted antioxidant nanotherapies have found success in pre-clinical studies. SUMMARY: This review underscores the potential of targeted nanocarriers as means of finding success translating antioxidant therapies to the clinic, all with a focus on CVD.

Compression force sensing regulates integrin αIIbß3 adhesive function on diabetic platelets.

Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin αIIbß3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to αIIbß3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.

Thromboinflammatory Functions of Platelets in Ischemia-Reperfusion Injury and Its Dysregulation in Diabetes.

Ischemia-reperfusion (IR) injury is a common complication of a variety of cardiovascular diseases, including ischemic stroke and myocardial infarction (MI). While timely re-establishment of blood flow in a thrombosed artery is the primary goal of acute therapy in these diseases, paradoxically, reperfusion of ischemic tissue can cause widespread microvascular dysfunction that significantly exacerbates organ damage. Reperfusion injury is associated with activation of the humoral and cellular components of the hemostatic and innate immune systems and also with excessive reactive oxygen species production, endothelial dysfunction, thrombosis, and inflammation. Platelets are critical mediators of thromboinflammation during reperfusion injury and a hyperactive platelet phenotype may contribute to an exaggerated IR injury response. This is particularly relevant to diabetes which is characteristically associated with hyperactive platelets, significantly worse IR injury, increased organ damage, and increased risk of death. However, the mechanisms underlying vulnerability to IR injury in diabetic individuals is not well defined, nor the role of "diabetic platelets" in this process. This review summarizes recent progress in understanding the role of platelets in promoting microvascular dysfunction and inflammation in the context of IR injury. Furthermore, the authors discuss aspects of the thromboinflammatory function of platelets that are dysregulated in diabetes. They conclude that diabetes likely enhances the capacity of platelets to mediate microvascular thrombosis and inflammation during IR injury, which has potentially important implications for the future design of antiplatelet agents that can reduce microvascular dysfunction and inflammation.

Regulation of the nitric oxide oxidase activity of myeloperoxidase by pharmacological agents.

The leukocyte-derived heme enzyme myeloperoxidase (MPO) is released extracellularly during inflammation and impairs nitric oxide (NO) bioavailability by directly oxidizing NO or producing NO-consuming substrate radicals. Here, structurally diverse pharmacological agents with activities as MPO substrates/inhibitors or antioxidants were screened for their effects on MPO NO oxidase activity in human plasma and physiological model systems containing endogenous MPO substrates/antioxidants (tyrosine, urate, ascorbate). Hydrazide-based irreversible/reversible MPO inhibitors (4-ABAH, isoniazid) or the sickle cell anaemia drug, hydroxyurea, all promoted MPO NO oxidase activity. This involved the capacity of NO to antagonize MPO inhibition by hydrazide-derived radicals and/or the ability of drug-derived radicals to stimulate MPO turnover thereby increasing NO consumption by MPO redox intermediates or NO-consuming radicals. In contrast, the mechanism-based irreversible MPO inhibitor 2-thioxanthine, potently inhibited MPO turnover and NO consumption. Although the phenolics acetaminophen and resveratrol initially increased MPO turnover and NO consumption, they limited the overall extent of NO loss by rapidly depleting H2O2 and promoting the formation of ascorbyl radicals, which inefficiently consume NO. The vitamin E analogue trolox inhibited MPO NO oxidase activity in ascorbate-depleted fluids by scavenging NO-consuming tyrosyl and urate radicals. Tempol and related nitroxides decreased NO consumption in ascorbate-replete fluids by scavenging MPO-derived ascorbyl radicals. Indoles or apocynin yielded marginal effects. Kinetic analyses rationalized differences in drug activities and identified criteria for the improved inhibition of MPO NO oxidase activity. This study reveals that widely used agents have important implications for MPO NO oxidase activity under physiological conditions, highlighting new pharmacological strategies for preserving NO bioavailability during inflammation.

Mechanism and regulation of peroxidase-catalyzed nitric oxide consumption in physiological fluids: critical protective actions of ascorbate and thiocyanate.

Catalytic consumption of nitric oxide (NO) by myeloperoxidase and related peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how peroxidases consume NO in physiological fluids, in which multiple reactive enzyme substrates and antioxidants are present. Notably, ascorbate has been proposed to enhance myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by myeloperoxidase and related peroxidases (lactoperoxidase, horseradish peroxidase) by acting as a competitive substrate for protein-bound redox intermediates and by efficiently scavenging peroxidase-derived radicals (e.g., urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during inflammation. We show that NO consumption by myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However, thiocyanate effectively inhibits this process and yields hypothiocyanite at the expense of NO consumption. Hypothiocyanite can in turn form NO-consuming radicals, but thiols (albumin, glutathione) readily prevent this. Conversely, where ascorbate is absent, glutathione enhances NO consumption by urate radicals via pathways that yield S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating peroxidase-catalyzed NO consumption.