A New NO-Releasing Nanoformulation for the Treatment of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronicand progressive disease which continues to carry an unacceptablyhigh mortality and morbidity. The nitric oxide (NO) pathwayhas been implicated in the pathophysiology and progressionof the disease. Its extremely short half-life and systemiceffects have hampered the clinical use of NO in PAH. In anattempt to circumvent these major limitations, we have developeda new NO-nanomedicine formulation. The formulationwas based on hydrogel-like polymeric composite NO-releasingnanoparticles (NO-RP). The kinetics of NO release fromthe NO-RP showed a peak at about 120 min followed by asustained release for over 8 h. The NO-RP did not affect theviability or inflammation responses of endothelial cells. TheNO-RP produced concentration-dependent relaxations of pulmonaryarteries in mice with PAH induced by hypoxia. Inconclusion, NO-RP drugs could considerably enhance thetherapeutic potential of NO therapy for PAH

Activation and contraction of human ‘vascular’ smooth muscle cells grown from circulating blood progenitors

Blood outgrowth smooth muscle cells offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering and personalised medicine. However, little is known about these cells which has meant their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of blood outgrowth smooth muscle cells and vessel derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10 26 -6 M) increased cytosolic calcium in blood outgrowth smooth muscle cells fibroblasts. Increased calcium signal peaked between 10-20 seconds after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both blood outgrowth smooth muscle cells and vascular smooth muscle cells but not in fibroblasts. In summary, we show that functional responses of blood outgrowth smooth muscle cells are in line with vascular smooth muscle cells providing critical evidence of their application in biomedical research

Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways

Rationale Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known. Objective Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity. Methods and measurements 34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay. Main results Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis. Conclusions In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo

Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine : novel explanation of cardiovascular side effects associated with anti-inflammatory drugs

© 2014 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.BACKGROUND: Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified.METHODS AND RESULTS: Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage.CONCLUSIONS: We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.Peer reviewedFinal Published versio

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension

Aims. RhoB plays a key role in the pathogenesis of hypoxia - induced pulmonary hypertension. Farne sylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and Results. The analysis of l ung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia - induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia - induced vascular cell proliferation, increased endothelium - dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerisation and increased eNOS expression in vi tro and in vivo . Farnesylated - only RhoB (F - RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated - only RhoB (GG - RhoB) and tipifarnib had an inhibitory effect. Label - fre e proteomics linked F - RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F - RhoB - regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins. Conclusions. Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodeling and provides a rationale for selective targeting of this pa thway in pulmonary hypertension

A New NO-Releasing Nanoformulation for the Treatment of Pulmonary Arterial Hypertension

Copyright The Author(s) 2016. This article is published with open access at Springerlink.com. Open Access - This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were madePulmonary arterial hypertension (PAH) is a chronic and progressive disease which continues to carry an unacceptably high mortality and morbidity. The nitric oxide (NO) pathway has been implicated in the pathophysiology and progression of the disease. Its extremely short half-life and systemic effects have hampered the clinical use of NO in PAH. In an attempt to circumvent these major limitations, we have developed a new NO-nanomedicine formulation. The formulation was based on hydrogel-like polymeric composite NO-releasing nanoparticles (NO-RP). The kinetics of NO release from the NO-RP showed a peak at about 120 min followed by a sustained release for over 8 h. The NO-RP did not affect the viability or inflammation responses of endothelial cells. The NO-RP produced concentration-dependent relaxations of pulmonary arteries in mice with PAH induced by hypoxia. In conclusion, NO-RP drugs could considerably enhance the therapeutic potential of NO therapy for PAH.Peer reviewedFinal Published versio

A persistent neutrophil-associated immune signature characterizes post-COVID-19 pulmonary sequelae

Interstitial lung disease and associated fibrosis occur in a proportion of individuals who have recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through unknown mechanisms. We studied individuals with severe coronavirus disease 2019 (COVID-19) after recovery from acute illness. Individuals with evidence of interstitial lung changes at 3 to 6 months after recovery had an up-regulated neutrophil-associated immune signature including increased chemokines, proteases, and markers of neutrophil extracellular traps that were detectable in the blood. Similar pathways were enriched in the upper airway with a concomitant increase in antiviral type I interferon signaling. Interaction analysis of the peripheral phosphoproteome identified enriched kinases critical for neutrophil inflammatory pathways. Evaluation of these individuals at 12 months after recovery indicated that a subset of the individuals had not yet achieved full normalization of radiological and functional changes. These data provide insight into mechanisms driving development of pulmonary sequelae during and after COVID-19 and provide a rational basis for development of targeted approaches to prevent long-term complications

Cyclooxygenases and the cardiovascular system.

Cyclooxygenase (COX)-1 and COX-2 are centrally important enzymes within the cardiovascular system with a range of diverse, sometimes opposing, functions. Through the production of thromboxane, COX in platelets is a pro-thrombotic enzyme. By contrast, through the production of prostacyclin, COX in endothelial cells is antithrombotic and in the kidney regulates renal function and blood pressure. Drug inhibition of COX within the cardiovascular system is important for both therapeutic intervention with low dose aspirin and for the manifestation of side effects caused by nonsteroidal anti-inflammatory drugs. This review focuses on the role that COX enzymes and drugs that act on COX pathways have within the cardiovascular system and provides an in-depth resource covering COX biology and pharmacology. The review goes on to consider the role of COX in both discrete cardiovascular locations and in associated organs that contribute to cardiovascular health. We discuss the importance of, and strategies to manipulate the thromboxane: prostacyclin balance. Finally within this review the authors discuss testable COX-2-hypotheses intended to stimulate debate and facilitate future research and therapeutic opportunities within the field

The role of nitric oxide synthase inhibitors on macrophage function

Asymmetrically methylated forms of arginine (asymmetric dimethylarginine, ADMA and L-N-monomethylarginine, L-NMMA) are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). These molecules are produced endogenously in all cells by a process that involves protein arginine methyltransferase (PRMT)-catalysed methylation of certain arginine residues in proteins and the subsequent proteolysis of these methylated proteins. Free methylarginines accumulate in the cytosol where they are actively metabolized to citrulline and methylamines by the enzyme dimethylarginine dimethylaminohydrolase (DDAH1 and 2). ADMA and L-NMMA have been shown to be elevated in patients with cardiovascular and renal disease, however, their role in immune cells and in particular macrophages is yet to be confirmed. The aim of this project was to determine the effect of increased ADMA, both endogenous and exogenous, on macrophage function. U937-derived macrophages (a human monocytic cell line) and primary peritoneal macrophages extracted from wild type (DDAH2+/+), global DDAH2 knock-out (DDAH2-/-) and macrophage-specific DDAH2 knock-out (DDAH2flox/flox LysM-Cre) mice were used. Their NO production, motility, phagocytosis, chemotaxis, and cytokine levels was determined using the Griess assay, real-time imaging, a fluorescently labelled E-coli phagocytosis assay, a transwell migration assay and RNA-sequencing respectively. Here we show that, in contrast to endothelial cells, DDAH2 is the only DDAH isoform expressed in primary murine macrophages basally or following cytokine stimulation of the cells. Results showed that DDAH2 metabolises methylarginines in macrophages through a VEGF-independent mechanism and significantly attenuates cytokine-stimulated NO synthesis. Both the pharmacological addition of ADMA and genetic deletion of DDAH2 in macrophages results in impaired macrophage function (as assessed by motility, phagocytosis and cytokine production). These data identify DDAH2 as a key regulator of macrophage NO synthesis and demonstrate the potential therapeutic utility of DDAH2-selective inhibitors

Incidence of haematological malignancies in Kosovo-A post "uranium war" concern.

BACKGROUND:During the Kosovo War (1998-99) approximately 31,000 rounds with Depleted Uranium (DU) were fired on 85 targets in Kosovo. The number of haematological malignancies (HM) increased after the war and the concern was the use of DU during the war. The aim of this study was to analyse the incidence rates of HM in Kosovo throughout a 20-year that includes pre- and post- war period (1995-2015); and to examine if there is any association between the use of DU rounds and incidence rates of HM in different regions of Kosovo. METHODS:In this retrospective register-based study, 1,798 new patients diagnosed with leukaemia, Hodgkin lymphoma, non-Hodgkin lymphoma and Multiple myeloma were analysed over a 20 year period. Incidence rates were calculated focusing on specific time periods, regions and age-groups. In addition, the correlation between the use of DU in different regions and their incidence of HM was analysed. RESULTS:The average annual crude rate of all HM in Kosovo was 5.02 cases per 100,000 persons. Incidence rates of HM in first post-war period (2000-2003) increased by 0.37 cases/100,000 persons (9.51%) compared to the pre-war period (1995-1998) whereas in the last post-war period (2012-2015), incidence of HM increased by 3.19/100,000 persons (82%). Gjakova and Peja, the first and third most exposed regions to DU ordnance ranked first and second in difference in HM. Prishtina, Gjilan and Ferizaj, regions with the least number of rounds/km2, were characterized by a decline of incidence rates. CONCLUSIONS:After the war, the increase in incidence rate of HM was higher in two regions with most DU rounds/km2 expended Despite these findings, this study warrants further investigation and does not lead us to a conclusive finding on the existence of a causal relationship between the use of DU during the war and the rise in incidence of HM in Kosovo