MELD-score for risk stratification in cardiac surgery.

The outcome of the patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) is also influenced by the renal and hepatic organ functions. Risk stratification, using scores such as EURO Score II or STS Short-Term Risk Calculator for patients undergoing cardiac surgery with cardiopulmonary bypass, ignores the quantitative renal and hepatic function; therefore, MELD-Score was applied in these cases. We retrospectively examined patient data using the MELD score as a predictor of mortality. To perform a univariate analysis of the data, patients were classified into three groups based on the MELD Score: MELD < 10 (Group 1), MELD 10 to 19 (Group 2), and MELD ≥ 20 (Group 3). A total of 11,477 participants were included in the study, though several patients with either missing MELD scores or lack of creatinine, bilirubin, or INR levels were dropped from the original cohort. Eventually, 10,882 patients were included in the analysis. The primary outcome was defined as postoperative, in-hospital mortality. Secondary outcomes such as postoperative bleeding, including the requirement for repeat thoracotomy, postoperative neurological complications, and assessment of catecholamines on weaning from cardiopulmonary bypass/ requirement of mechanical circulatory support were examined. A higher MELD score was associated with increased postoperative mortality. Patients with MELD > 20 experienced a 31.2% postoperative mortality, compared to Group 1 (4.6%) and Group 2 (17.5%). The highest rates of postoperative bleeding (13.8%) and, repeat thoracotomy (13.2%) & postoperative pneumonia (17.4%) were seen in Group 3 (threefold increase when compared to Group 1, renal failure requiring dialysis (N = 235, 2.7% in Group 1 v/s N = 78, 22.9% in Group 3) or requiring high dose catecholamines post-operatively or mechanical circulatory support (IABP/ECLS). Incidentally, an increased MELD Score was not associated with a significant increase in the postoperative incidence of stroke/TIA or the presence of sternal wound infections/complications. A higher mortality was observed in patients with reduced liver and renal function, with a significant increase in patients with a MELD score > 20. As the current risk stratification scores do not consider this, we recommend applying the MELD score before considering patients for cardiac surgery.

Wearable cardioverter defibrillator after cardiac surgery: Analysis of real-life data from patients at transient risk of sudden cardiac death.

BACKGROUND: Especially in the first 3 months after cardiac surgery, patients are at transient risk of sudden cardiac death (SCD). To close the gap between hospital discharge and the final implantable cardioverter-defibrillator (ICD) decision, guidelines recommend temporarily using a wearable cardioverter-defibrillator (WCD) to protect these patients from SCD. We investigated real-life data on the safety, effectiveness, and compliance of the WCD in this population. METHODS: Data for analysis were collected via the Zoll Patient Management Network (ZPM) from patients who underwent cardiac surgery and who were discharged with a WCD between 2018 and 2021 at the Cardiac Surgery Center of the University of Erlangen in Germany. RESULTS: The majority of the 55 patients were male (90.9%) and underwent a coronary artery bypass graft (80.0%). The number of patients with left ventricular ejection fraction (LVEF) >35% increased from 9.1% at the beginning of WCD use to 58.2% at the end of WCD use. Six ventricular tachycardia (VT) episodes occurred in four patients. The WCD appropriately defibrillated two patients with VT episodes. There were no inadequate shocks and no fatalities during the observation time. WCD wearing compliance was high, with a median wear time of 23.3 h/day. CONCLUSION: This retrospective analysis in a single cardiac surgery center confirms prior data on the safety and effectiveness of the WCD in patients in post-surgery care in a real-life setting. The WCD successfully protected patients from SCD during life-threatening VT episodes. WCD wearing compliance was high.

Single-Center Retrospective Analysis of Acute Type A Aortic Dissection Outcome and Reoperation Focusing on Extended Versus Limited Initial Repair.

BACKGROUND: The optimal management strategy for acute aortic type A dissection remains controversial. Whether a limited primary (index) repair would increase the need for late aortic reintervention is still an open debate. METHODS: A total of 393 consecutive adult patients with acute type A aortic dissection who underwent cardiac surgery were analyzed. Our research hypothesis was whether limited aortic index repair (i.e., isolated aorta ascending replacement without an open distal anastomosis with and without a concomitant aortic valve replacement, including hemiarch replacement procedure) is associated with a higher incidence of late aortic reoperation compared with extended repair (i.e., any other surgical procedure that goes beyond that limited approach). RESULTS: Type of the initial repair had no statically significant relationship with in-hospital mortality with a P-value of 0.12, however in multivariable analysis, cross-clamp time had a statistically significant relation with mortality (P = 0.4). From the patients who survived until discharge (N = 311), 40 patients needed a reoperation on the aorta; the mean interval until reoperation was 4.5 years. The relationship between the type of the initial repair and the need for reoperation didn't reach a statically significant value (P = 0.9). In-hospitable mortality after the second operation was 10% (N = 4). CONCLUSION: We reached two conclusions. 1) An extended prophylactic repair in the initial operation of an acute type A aortic dissection might not lead to a lower incidence of reoperations on the aorta and could increase in-hospital mortality by increasing cross-clamp time, and 2) Reoperation on the aorta could be done safely with acceptable mortality outcomes.

Pass On What You Have Learned: A Structured Mentor-Mentee Concept for the Implementation of a Minimally Invasive Mitral Valve Surgery Program.

INTRODUCTION: Starting a minimally invasive cardiac surgery (MICS) for mitral valve repair (MVR) program is challenging as it requires a new learning curve, but compromising surgical results at the same time is not acceptable. Here, we describe our surgical educational experience of starting a new MICS program at a university heart center in Germany. METHODS: A dedicated team for the new MICS program including 2 cardiac surgeons, 1 cardiac anesthetist, 1 perfusionist, and 1 scrub nurse was chosen. The use of long shafted instruments was trained in a low-cost self-assembled MICS simulator, and the EACTS endoscopic dry lab course was visited. Thereafter, 1 MICS center was visited for direct observation and peer-to-peer education for 6 weeks. The mentor observed the first 10 cases performed by the mentee. The surgical mitral valve expertise of 1 single cardiac surgeon was retrospectively analyzed between April 2016 and April 2021. RESULTS: Before the implementation of the MICS-MVR program, 18 mitral valve operations have been performed through sternotomy between April 2016 and October 2018 including 12 replacements and 6 ring annuloplasties. After starting the MICS-MVR program, 73 mitral operations have been performed by the same surgeon of which 53 video-assisted through minithoracotomy (72.6%). 83.1% of the MICS procedures included complex repair (n = 38) and ring annuloplasty (n = 6). Open heart MV surgery was necessary in 20 patients due to concomitant procedures (n = 8), redo procedures (n = 2), severe endocarditis (n = 4), or contraindication for MICS such as PAD (n = 6). There have been no deaths, 1 stroke, and 1 cardiac vascular (RCX) complication. Two patients required conversion to sternotomy and one pericardiocentesis in the long term. CONCLUSION: Typically, excellent exposure and high repair rates of the MV has led us offer MICS approach to a majority of patients with isolated MV disease. Careful planning and a strict mentor-mentee concept facilitated a safe startup of an MICS program in a busy university heart center.

Parasite infection influences the biomarker response and locomotor activity of Gammarus fossarum exposed to conventionally-treated wastewater.

Modern wastewater treatment plants cannot completely remove pollutants. Often, effluents entering the aquatic environment still contain micropollutants such as pharmaceuticals or pesticides, which may impose adverse effects on aquatic biota. At the same time, a large proportion of free-living aquatic species are known to be infected with parasites, which raises the question of interactions between environmental stressors (such as micropollutants) and parasite infection. We chose the freshwater amphipod Gammarus fossarum (Koch, 1835) as a test organism to investigate potential pollutant-parasite interactions. This gammarid is frequently used in ecotoxicological tests and is also commonly infected with larvae of the acanthocephalan parasite species Polymorphus minutus (Zeder, 1800) Lühe, 1911. We exposed infected and uninfected specimens of G. fossarum to conventionally-treated wastewater and river water in a 22-day flow channel experiment. The test organisms' response was measured as mortality rates, concentrations or activities of five biomarkers, and overall locomotor activity. No significant differences were found between mortality rates of different exposure conditions. Contrastingly, three biomarkers (phenoloxidase activity, glycogen, and lipid concentrations) showed a significant increase in infected gammarids, while the effect of the water type was insignificant. Infected gammarids also showed a significantly higher locomotor activity in both water types. Our results suggest that the response of G. fossarum during the exposure experiments was mainly driven by parasite infection. This implies that parasites may act as additional biotic stressors in multiple stressor scenarios, and therefore, might play an important role when measuring the response of organisms to chemical stressors. Future ecotoxicological studies and assessments thus should consider parasite infection as an additional test parameter.

Recovery of a dilated left ventricle after cessation of cocaine and HVAD™ explantation using a titanium plug.

The ultimate goal in the treatment of end-stage heart failure is the recovery of cardiac function following mechanical assistance of the left ventricle. The HVAD™ pump (HeartWare Inc.) left ventricular assist device (LVAD) can be explanted without resternotomy. This article demonstrates that the use of a custom-made mechanical plug (manufactured by INNOVO Solutions GmbH), which can be inserted into the LVAD's sewing ring, is feasible. This mechanical plug explicitly designed for device explantation is a viable alternative to the current standard of care. This article adopts a less invasive technique to explant the pump. The following case illustrates this technique.

Successive surgical repair of a progressive aortic dissection in a case of Loeys-Dietz Syndrome.

INTRODUCTION: The Frozen Elephant Trunk technique is a well-established treatment for aortic dissections (Stanford Type A) involving the aortic arch and descending aorta. The Thoraflex™ Hybrid prosthesis (Vascutek Ltd.), consisting of a proximal flexible conduit and a distal self-expanding covered stent, has consistently shown positive results in the treatment of this condition. CASE DESCRIPTION: The following is a description of such a staged reconstruction using the Thoraflex™ Hybrid Ante-Flo™ device, performed in a patient previously diagnosed with Loeys-Dietz Syndrome. After clamping the aorta proximally, an incision was taken at the distal end of the stent. Here, the distal end of the new prosthesis was inserted into the true lumen of the descending aorta and the stent was deployed. Following this, a bypass was established via the left atrium, and blood was returned to the lower body using the perfusion arm of the prosthesis with the proximal part of the descending aorta clamped. The collar of the prosthesis was sutured proximally to the aorta near the inlying previous stent. Air was removed via the perfusion arm of the prosthesis, which was then oversewn. DISCUSSION: To date, hybrid prostheses have only been used on the proximal aorta in patients with aortic aneurysms and aortic dissections (Stanford Type A). CONCLUSION: Using the described technique, it is possible to reconstruct the aortic anatomy using the available prostheses with accuracy and minimal complications.

Retrograde washout of prepump LVAD thrombosis in a patient on HeartWare™ support.

The success of the left ventricular assist device (LVAD) as a treatment for terminal left-side heart failure is still restrained by some severe complications associated with mechanical circulatory support. Pump thrombus still affects many patients. It is associated with high morbidity and mortality. The therapeutic options include augmentation of anticoagulation and antiplatelet medication, intravenous or catheter-guided thrombolysis, and pump exchange. Heart transplantation would be a desirable option in this population, but unfortunately, it is only theoretical given the increasing number of LVAD implants and decreasing number of organ donors. A retrograde washout maneuver may be a treatment option in prepump thrombosis in selected patients. Therefore, the decision should be made on an individual basis after balancing the risks and benefits of different treatment approaches. In this context, we report a case of retrograde washout of prepump thrombus in a patient who has been on HeartWare™ support for more than 3 years, with a successful bailout strategy.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.

Crystal structure of a class III adenylyl cyclase-like ATP-binding protein from Pseudomonas aeruginosa.

In many organisms, the ubiquitous second messenger cAMP is formed by at least one member of the adenylyl cyclase (AC) Class III. These ACs feature a conserved dimeric catalytic core architecture, either through homodimerization or through pseudo-heterodimerization of a tandem of two homologous catalytic domains, C1 and C2, on a single protein chain. The symmetric core features two active sites, but in the C1-C2 tandem one site degenerated into a regulatory center. Analyzing bacterial AC sequences, we identified a Pseudomonas aeruginosa AC-like protein (PaAClp) that shows a surprising swap of the catalytic domains, resulting in an unusual C2-C1 arrangement. We cloned and recombinantly produced PaAClp. The protein bound nucleotides but showed no AC or guanylyl cyclase activity, even in presence of a variety of stimulating ligands of other ACs. Solving the crystal structure of PaAClp revealed an overall structure resembling active class III ACs but pronounced shifts of essential catalytic residues and structural elements. The structure contains a tightly bound ATP, but in a binding mode not suitable for cAMP formation or ATP hydrolysis, suggesting that PaAClp acts as an ATP-binding protein.

Pericardectomy after pericarditis constrictiva led to onset of transplant kidney function after 98 days of anuric kidney graft: a case report.

BACKGROUND: Constrictive pericarditis is easily overlooked and can lead to severe problems in hemodynamics and end-organ perfusion, in our patient leading to 98 days of anuria after living kidney transplantation. This was completely reversible after pericardectomy. CASE PRESENTATION: A 43-year-old female caucasian patient received a living kidney donation from her mother. She had developed end-stage renal disease 2 years prior due to nephrotic syndrome linked to graft-versus-host disease after allogenic stem-cell transplantation for aplastic anemia. The graft showed insufficient function already in the early postoperative phase. Dialysis was paused after surgery, but the patient developed hypervolemia with ascites and edema in the lower extremities. Doppler ultrasonography showed scarce perfusion, with intrarenal arterial waveforms without end-diastolic flow. The venous perfusion profiles showed pulsatile retrograde flow. There was no identifiable reason for a primary vascular perfusion problem on ultrasonography or transplant kidney angiography. Kidney transplant biopsy revealed no rejection but extensive acute tubular necrosis. Three weeks after transplantation, the patient developed an acute anuric graft failure caused by severe cardiac decompensation. Echocardiography revealed a previously unnoticed constrictive pericarditis, which could be confirmed in a cardio computed tomography scan. The constrictive pericarditis had not been apparent on previous x-rays, computed tomography scans, or echocardiographies, including those for transplantation evaluation. Conservative management of the constrictive pericarditis was not successful and the graft remained anuric. Eventually, the patient underwent pericardectomy 16 weeks after kidney transplantation. Shortly after surgery, the graft started urine production again, which significantly increased within a few days. The clearance improved and 2 weeks later, the patient was free from dialysis. CONCLUSIONS: This case illustrates that special attention should be given to the pericardium during transplant evaluation, especially for patients who previously underwent stem-cell transplantations, chemotherapy or radiation.

Glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux.

A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte transverse tubular system (t-system), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-system is altered in GRKO hearts and if GR signalling is required for t-system preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-system density and increased distances between ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC). Because t-system remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-system density than cells treated with dexamethasone (EC50 1.1 nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR-LTCC coupling and synchrony of intracellular Ca2+ release, but did not alter expression levels of t-system-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-system preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-system loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-system. These findings suggest that GR signalling and autophagy are critically involved in t-system preservation and remodelling in the heart.

DJ1 and microRNA-214 act synergistically to rescue myoblast cells after ischemia/reperfusion injury.

Ischemia/reperfusion injury is a tissue injury occurring post-reperfusion of tissues with pre-existing ischemia. A good blood supply to tissues aids in the survival of ischemic tissue, however, due to prolonged ischemia the levels of ATP decrease and pH declines leading to acidosis. Reduced ATP leads to an increase in the AMP/ATP ratio, causing cessation of intracellular calcium transport, hence calcium overload and cell death. In this study, we demonstrate the synergistic and antagonistic effect of DJ1 and microR-214 (miR-214) in rescuing myoblast C2C12 cells after ischemia/reperfusion in an in vitro model. Both DJ1 and miR-214 were cloned into a hypoxic inducible expression cassette and transfected into the C2C12 cells. We showed that DJ1 and miR-214 have synergistic effects in reducing intracellular lactate dehydrogenase and intracellular transient calcium levels after reoxygenation compared to control cells, in addition to reducing cell death via necrosis. Western blotting revealed a decrease in autophagosome formation in LC3II/I ratio and an increase in AKT expression in cells transfected with DJ1 and miR-214. Using quantitative real-time PCR, we demonstrated that DJ1 and miR-214 significantly reduced the expression of pro-apoptotic factors and autophagy compared to control. The results indicated DJ1 is an endogenous oxidative stress molecule and miR-214 is a potent inhibitor of the sodium calcium exchanger channel. DJ1 had the greatest effect to inhibiting mitochondrial cell death pathways by possibly acting as a modulator of autophagy. Additionally, we have concluded that miR-214 has an inhibitory effect on extrinsic cell death pathways such as necrosis and autophagy.

New Targets for the Prevention of Chronic Rejection after Thoracic Organ Transplantation.

The gold standard for the treatment of terminal heart failure and irreversible lung diseases includes thoracic organ transplantation. The major obstacle for long-term survival after successful transplantation is chronic rejection, an ongoing immunomodulatory disease so far without effective therapy. Therefore, the aim of this review is to elucidate scientific efforts targeting different new mechanisms of cardiac allograft vasculopathy (CAV) and chronic lung allograft dysfunction (CLAD). For this purpose, we performed a systematic review of the literature to assess recent strategies in transplant immunology research. We searched MEDLINE from 2015 up to date for articles addressing the following keywords: CAV, transplant vasculopathy, transplant arteriosclerosis, CLAD, bronchiolitis obliterans transplant, and obliterative bronchiolitis transplant. All articles including experimental models in the field of transplant immunology addressing new aspects for the prevention of chronic rejection after heart and lung transplantation were included in this review. The prevention of chronic rejection would clearly improve the survival of patients after heart and lung transplantation. Interesting targets were addressed in recent research, but further research is necessary to effectively treat this life-threatening disease in transplant recipients.

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate.

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.

Synthetic Glycoforms Reveal Carbohydrate-Dependent Bioactivity of Human Saposin†D.

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.

Clopidogrel significantly lowers the development of atherosclerosis in ApoE-deficient mice in vivo.

The anti-platelet drug clopidogrel has been shown to modulate adhesion molecule and cytokine expression, both playing an important role in the pathogenesis of atherosclerosis. The aim of this study was to investigate the impact of clopidogrel on the development and progression of atherosclerosis. ApoE(-/-) mice fed an atherogenic diet (cholesterol: 1 %) for 6 months received a daily dose of clopidogrel (1 mg/kg) by i.p. injection. Anti-platelet treatment was started immediately in one experimental group, and in another group clopidogrel was started 2 month after beginning of the atherogenic diet. Blood was analysed at days 30, 60 and 120 to monitor the lipid profile. After 6 months the aortic arch and brachiocephalic artery were analysed by Sudan IV staining for plaque size and by morphometry for luminal occlusion. Serum levels of various adhesion molecules were investigated by ELISA and the cellular infiltrate was analysed by immunofluorescence. After daily treatment with 1 mg/kg clopidogrel mice showed a significant reduction of atherosclerotic lesions in the thoracic aorta and within cross sections of the aortic arch [plaque formation 55.2 % (clopidogrel/start) vs. 76.5 % (untreated control) n = 8, P < 0.05]. After treatment with clopidogrel P-/E-selectin levels and cytokine levels of MCP-1 and PDGFß were significantly reduced as compared to controls. The cellular infiltrate showed significantly reduced macrophage and T-cell infiltration in clopidogrel-treated animals. These results show that clopidogrel can effectively delay the development and progression of 'de-novo' atherosclerosis. However, once atherosclerotic lesions were already present, anti-platelet treatment alone did not result in reverse remodelling of these lesions.

Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism.

Sirtuins are protein deacetylases regulating metabolism and stress responses. The seven human Sirtuins (Sirt1-7) are attractive drug targets, but Sirtuin inhibition mechanisms are mostly unidentified. We report the molecular mechanism of Sirtuin inhibition by 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (Ex-527). Inhibitor binding to potently inhibited Sirt1 and Thermotoga maritima Sir2 and to moderately inhibited Sirt3 requires NAD(+), alone or together with acetylpeptide. Crystal structures of several Sirtuin inhibitor complexes show that Ex-527 occupies the nicotinamide site and a neighboring pocket and contacts the ribose of NAD(+) or of the coproduct 2'-O-acetyl-ADP ribose. Complex structures with native alkylimidate and thio-analog support its catalytic relevance and show, together with biochemical assays, that only the coproduct complex is relevant for inhibition by Ex-527, which stabilizes the closed enzyme conformation preventing product release. Ex-527 inhibition thus exploits Sirtuin catalysis, and kinetic isoform differences explain its selectivity. Our results provide insights in Sirtuin catalysis and inhibition with important implications for drug development.