Immunoglobulins Against Tyrosine-Nitrated Epitopes in Coronary Artery Disease

Background—Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results—Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 μg antibody Eq/mL plasma versus 0.36±0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions—The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD

Extracellular post-translational modifications of proteins in experimental models of atherosclerosis

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and anti-inflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA–apoA-I⁻/⁻). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA–apoA-I⁻/⁻ as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the atherosclerotic lesions of LA–apoA-I⁻/⁻ as compared with the LA mice. Proteomic approaches based on affinity enrichment and site specific adduct mapping identified 44 proteins in the atherosclerotic lesions and 17 proteins in the plasma of LA–apoA-I⁻/⁻ models. In particular, the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in the plasma of LA–apoA-I⁻/⁻ as compared with the LA mice. Furthermore, we evaluated the role of fibrinogen nitration on the development of thrombotic complications in humans. Previous data demonstrated elevated levels of nitrated fibrinogen in subjects with coronary artery disease, and accelerated clotting when fibrinogen was nitrated in vitro. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Utilizing state of the art proteomic approaches we identified the tyrosine residues 292 and 422 at the carboxyl terminus of the β-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob “B” mimetic peptides Gly-His-Arg-Proam and AlaHis-Arg-Proam suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.Η απολιποπρωτεΐνη Α-Ι, η βασική πρωτεΐνη εντός του µορίου της υψηλής πυκνότητας χοληστερόλης (HDL), έχει συσχετισθεί µε αθηροπροστατευτικές ιδιότητες µέσω του µηχανισµού της ανάδροµης µεταφοράς χοληστερόλης από την περιφέρεια προς το ήπαρ. Ωστόσο, αντιφλεγµονώδεις και αντιοξειδωτικές ιδιότητες έχουν επίσης προταθεί. Στην παρούσα εργασία µελετήθηκαν οι αντιοξειδωτικές ιδιότητες της απολιποπρωτεΐνης Α-Ι µε την ανίχνευση και ποσοτική µέτρηση των επιπέδων νιτροτυροσίνης, τόσο στις αορτικές ρίζες, όσο και στο πλάσµα αθηροσκληρωτικών µοντέλων µυών τύπου LDLr⁻/⁻ υπό την παρουσία (µοντέλα LA) ή απουσία (µοντέλα LA–apoA-I⁻/⁻) της απολιποπρωτεΐνης Α-Ι. Τα επίπεδα νιτροτυροσίνης στις αορτικές ρίζες των µοντέλων LA–apoA-I⁻/⁻, βρέθηκαν 6 φορές υψηλότερα συγκριτικά µε τα µοντέλα LA. Η παρατήρηση αυτή επαληθεύθηκε µε τη χρήση µεθόδων ανοσοϊστοχηµείας και ανοσοηλεκτρονικής µικροσκοπίας, τα οποία έδειξαν εντονότερη χρώση για νιτροτυροσίνη στις αθηρωµατικές αλλοιώσεις των µοντέλων που δεν εκφράζουν την απολιποπρωτεΐνη Α-Ι. Επιπρόσθετα, µε την εφαρµογή της τεχνολογίας της πρωτεοµικής, ανιχνεύθηκαν 44 πρωτεΐνες - στόχοι προς νίτρωση στις αθηρωµατικές πλάκες και 17 πρωτεΐνες - στόχοι προς νίτρωση στο πλάσµα των µοντέλων LA–apoA-I⁻/⁻. Το ινωδογόνο βρέθηκε νιτρωµένο τόσο εντός του πλάσµατος, όσο και µέσα στο αθήρωµα. Επειδή σε προηγούµενες εργασίες βρέθηκε ότι η νίτρωση του ινωδογόνου in vitro οδηγεί σε επιταχυνόµενη δηµιουργία θρόµβου, υποθέσαµε πως η µετα-µεταφραστική τροποποίηση αυτού µε νίτρωση in vivo µπορεί να προκαλεί αυξηµένη τάση για θροµβωτικά επεισόδια. Οι λειτουργικές επιπτώσεις της νίτρωσης µελετήθηκαν σε δείγµατα ινωδογόνου που αποµονώθηκαν από το πλάσµα υγιών ατόµων. Με την εφαρµογή της πρωτεοµικής αναγνωρίσθηκαν τα υπολείµµατα τυροσίνης 292 και 422 στο καρβοξυτελικό άκρο της β-αλύσου του µορίου του ινωδογόνου ως οι κύριοι στόχοι προς νίτρωση in vivo. Πειράµατα µε ανοσοηλεκτρονική µικροσκοπία απέδειξαν ότι τα νιτρωµένα µόρια πολυµερίζονται και ενσωµατώνονται στο σχηµατιζόµενο θρόµβο. Αποδείξαµε ότι η νίτρωση του ινωδογόνου προκαλεί 4 λειτουργικές επιπτώσεις: i) αύξηση της ταχύτητας πολυµερισµού, ii) µεταβολή της αρχιτεκτονικής του θρόµβου ινικής, iii) αύξηση της σκληρότητας και ελάττωση της πλαστικότητας του θρόµβου και iv) ελάττωση της ταχύτητας ινωδόλυσης από το ένζυµο πλασµίνη. Στα πειράµατα ελέγχου η εκλεκτική αφαίρεση των νιτρωµένων µορίων είχε ως αποτέλεσµα την ελάττωση της ταχύτητας πολυµερισµού και την εξάλειψη των µεταβολών στην αρχιτεκτονική του θρόµβου. Επιπρόσθετα πειράµατα µε τη χρήση των µιµητικών πεπτιδίων των κοµβίων “B” Gly-His-ArgProam και Ala-His-Arg-Proam υποδηλώνουν πως τα νιτρωµένα µόρια οδηγούν στις παραπάνω λειτουργικές επιπτώσεις µέσω της επιτάχυνσης της πλάγιας συγκόλλησης των ινών ινικής. Τα δεδοµένα αυτά αναδεικνύουν έναν νέο παράγοντα κινδύνου, το νιτρωµένο ινωδογόνο, που εξηγεί την επιδηµιολογική συσχέτιση µεταξύ οξειδωτικού stress και αθηροθροµβωτικών επεισοδίων

Sex differences in corticotropinreleasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology

Although the higher incidence of stress-related psychiatric disorders in females is well documented, its basis is unknown. Here we demonstrate that the receptor for corticotropin-releasing factor (CRF), the neuropeptide that orchestrates the stress response, signals and is trafficked differently in female rats in a manner that could result in a greater response and decreased adaptation to stressors. Most cellular responses to CRF in the brain are mediated by CRF receptor (CRFr) association with the GTP-binding protein, Gs. Receptor immunoprecipitation studies revealed enhanced CRFr-Gs coupling in cortical tissue of unstressed female rats. Previous stressor exposure abolished this sex difference by increasing CRFr-Gs coupling selectively in males. These molecular results mirrored the effects of sex and stress on sensitivity of locus ceruleus (LC)-norepinephrine neurons to CRF. Differences in CRFr trafficking were also identified that could compromise stress adaptation in females. Specifically, stress-induced CRFr association with β-arrestin2, an integral step in receptor internalization, occurred only in male rats. Immunoelectron microscopy confirmed that stress elicited CRFr internalization in LC neurons of male rats exclusively, consistent with reported electrophysiological evidence fo

Mean Aortic pressure gradient and global longitudinal strain recovery after transcatheter aortic valve replacement – A retrospective analysis

Background: Global longitudinal strain (GLS) has incremental value in assessing left ventricular (LV) function in severe aortic stenosis and is related to clinical outcome after transcatheter aortic valve replacement (TAVR). We sought to identify relevant echocardiographic predictors of GLS improvement and myocardial function recovery after TAVR. Methods: We analyzed baseline and 12-month follow-up echocardiograms for LV strain analysis from 123 patients who underwent at Emory University Hospital with the Edwards SAPIEN valve between 7/2007 and 7/2013. Results: At baseline, 61 had reduced LV ejection fraction (LVEF) ≤50% (rEF), and 80 had preserved LVEF >50% (pEF). Higher baseline mean pressure gradient (MPG) and aortic peak velocity (AV Vmax) predicted myocardial function recovery defined as ≥20% improvement in global longitudinal strain (r = 0.29, p < .001; r = 0.26, p = .002). When analyzing subjects with discordant changes in GLS and LVEF at follow-up, subjects with improved GLS, although reduced LVEF after TAVR, experienced a greater reduction in MPG and AV Vmax (−40 vs. −30, p = 0.015; −2.3 vs. −1.9, p = .021) after the procedure. Conclusions: In high-risk patients undergoing TAVR for severe aortic stenosis, GLS is impaired and more impaired in patients with reduced EF. Higher baseline MPG predicts myocardial function recovery. GLS improvement after TAVR is related to relief of pressure overload. Keywords: Transcatheter aortic valve replacement (TAVR), Global longitudinal strain (GLS), Aortic stenosis, Stroke volume index, Subject codes: Aortic Valve Replacement/Transcatheter Aortic Valve Implantation, Remodeling, Echocardiography, Valvular Heart Diseas

The role of multidetector CT scan in the management of prosthetic aortic valve thrombosis: A case report

Key Clinical Message In this case report, the utility of MDCT in elucidating the pathophysiology and etiology of prosthetic aortic valve dysfunction allowed us to distinguish thrombosis from pannus as an etiology of prosthetic valve dysfunction. MDCT also guided the success of therapy. Abstract The diagnosis and management of prosthetic aortic valve thrombosis (PAVT) is challenging. The accurate diagnosis of this entity and its prompt management is vital to improving the prognosis of PAVT patients. Multidetector CT plays a central role in this effort. We present a case of PAVT in which the use of MDCT was useful in guiding management