Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model

Anti-Gal antibodies; Bioprosthetic heart valves; Cellular immune infiltrateAnticuerpos anti-Gal; Válvulas cardíacas bioprotésicas; Infiltrado inmune celularAnticossos anti-Gal; Vàlvules cardíaques bioprotèsiques; Infiltrat immune cel·lularStructural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult α1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose α1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor.This research was funded by the European Union Seventh Framework Programme (FP7/2007–2013) under Grant Agreement no. 603049, TRANSLINK. This work was also supported by Ministerio de Economía y Competitividad-ISCiii (PI15/00181) and the PERIS SLT002/16/00445 funded by the Department of Health of Generalitat de Catalunya, all granted to CC and co-funded by FEDER funds, a way to build Europe. IDIBELL benefits from CERCA support. S.G.K. was partially supported by an IDIBELL summer internship. The funding agencies did not influence in any other way than by providing financial support

Integrated miRNA/mRNA Counter-Expression Analysis Highlights Oxidative Stress-Related Genes CCR7 and FOXO1 as Blood Markers of Coronary Arterial Disease

Our interest in the mechanisms of atherosclerosis progression (ATHp) has led to the recent identification of 13 miRNAs and 1285 mRNAs whose expression was altered during ATHp. Here, we deepen the functional relationship among these 13 miRNAs and genes associated to oxidative stress, a crucial step in the onset and progression of vascular disease. We first compiled a list of genes associated to the response to oxidative stress (Oxstress genes) by performing a reverse Gene Ontology analysis (rGO, from the GO terms to the genes) with the GO terms GO0006979, GO1902882, GO1902883 and GO1902884, which included a total of 417 unique Oxstress genes. Next, we identified 108 putative targets of the 13 miRNAs among these unique Oxstress genes, which were validated by an integrated miRNA/mRNA counter-expression analysis with the 1285 mRNAs that yielded 14 genes, Map2k1, Mapk1, Mapk9, Dapk1, Atp2a2, Gata4, Fos, Egfr, Foxo1, Ccr7, Vkorc1l1, Rnf7, Kcnh3, and Mgat3. GO enrichment analysis and a protein–protein-interaction network analysis (PPI) identified most of the validated Oxstress transcripts as components of signaling pathways, highlighting a role for MAP signaling in ATHp. Lastly, expression of these Oxstress transcripts was measured in PBMCs from patients suffering severe coronary artery disease, a serious consequence of ATHp. This allowed the identification of FOXO1 and CCR7 as blood markers downregulated in CAD. These results are discussed in the context of the interaction of the Oxstress transcripts with the ATHp-associated miRNAs

Chronic kidney disease-associated inflammation increases in risks of acute kidney injury and mortality after cardíac surgery

Cardiovascular mortality increases with decreasing renal function although the cause is yet unknown. Here, we have investigated whether low chronic inflammation in chronic kidney diseases (CKD) could contribute to increased risk for coronary artery diseases (CAD). Thus, a prospective case-control study was conducted in patients with CAD and CKD undergoing coronary artery bypass graft surgery with the aim of detecting differences in cardiovascular outcomes, epicardial adipose tissue volume, and inflammatory marker activity associated with renal dysfunction. Expression of membrane CD14 and CD16, inflammatory cytokines and chemokines, mitogen-activated protein (MAP) kinases and hsa-miR-30a-5p were analyzed in peripheral blood mononuclear cells (PBMCs). Epicardial fat volume and tissue inflammation in perivascular adipose tissue and in the aorta were also studied. In the present study, 151 patients were included, 110 with CAD (51 with CKD) and 41 nonCAD controls (15 with CKD). CKD increased the risk of cardiac surgery-associated acute kidney injury (CSA-AKI) as well as the 30-day mortality after cardiac surgery. Higher counts of CD14++CD16+ monocytes were associated with vascular inflammation, with an increased expression of IL1β, and with CKD in CAD patients. Expression of hsa-miR-30a-5p was correlated with hypertension. We conclude that CKD patients show an increased risk of CSA-AKI and mortality after cardiovascular surgery, associated with the expansion of the CD14++CD16+ subset of proinflammatory monocytes and with IL1β expression. We propose that inflammation associated with CKD may contribute to atherosclerosis (ATH) pathogenesis

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration

Outcomes research; Risk factorsInvestigación de resultados; Factores de riesgoRecerca dels resultats; Factors de riscBioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1–182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.This work was supported by the European Union Seventh Framework Program (FP7/2007/2013) under grant agreement no. 603049 for the Translink Consortium. This research was also funded by a European Union H2020 Program grant no. ERC-2016-STG-716220 to V.P-K. and by the Elizabeth and Nicholas Slezak Super Center for Cardiac Research and Medical Engineering (to V.P-K.). This work was supported by an Institut National de la Santé et de la Recherche Médicale translational grant no. 2012-2016 to T.L.T. This work was supported by the Ministerio de Economía y Competitividad-ISCiii (PI15/00181) and the PERIS SLT002/16/00445 funded by the Department of Health of Generalitat de Catalunya (both granted to C.C.), and cofunded by FEDER (European Regional Development Fund), a way to build Europe. This work was supported by an Israel Ministry of Science & Technology PhD fellowship to S.B. We thank L. Adler for her assistance in the affinity purification of anti-Neu5Gc antibodies and IgG subclass analysis. Finally, we thank N. Bovin from the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, who provided the Bdi-C3 PAA substrate needed to develop the anti-αGal assays

MiR-125b downregulates macrophage scavenger receptor type B1 and reverse cholesterol transport

Objective: To determine whether miR-125b regulates cholesterol efflux in vivo and in vitro through the regulation of scavenger receptor type B1 (SR-B1). Approach and results: We demonstrated that miR-125b is up-regulated in the human aortas of patients with CAD and is located in macrophages and vascular smooth muscle cells (VSMCs). We identified SCARB1 as a direct target of miR-125b by repressing the activity of the SCARB1 3'-untranslated region reporter construct. Moreover, the overexpression of miR-125b in both human and mouse macrophages as well as VSMCs was found to downregulated the expression of the SCARB1 and the SR-B1 protein levels, thereby impairing alpha-HDL-mediated macrophage cholesterol efflux in vitro. The in vivo reverse cholesterol transport (RCT) rate from non-cholesterol-loaded macrophages transfected with miR-125b to feces was also found to be decreased when compared with that of control mimic-transfected macrophages. Conclusions: Together, these results provide evidence that miR-125b downregulates SCARB1 and SR-B1 in both human and mouse macrophages as well as VSMCs, thereby impairing macrophage cholesterol efflux in vitro and the whole macrophage-specific RCT pathway in vivo

Response of the human myocardium to ischemic injury and preconditioning: The role of cardiac and comorbid conditions, medical treatment, and basal redox status

Vàlvula aòrtica; Isquèmia; MiocardiVálvula aórtica; Isquemia; MiocardioAortic valve; Ischemia; MyocardiumBackground The diseased human myocardium is highly susceptible to ischemia/reoxygenation (I/R)-induced injury but its response to protective interventions such as ischemic preconditioning (IPreC) is unclear. Cardiac and other pre-existing clinical conditions as well as previous or ongoing medical treatment may influence the myocardial response to I/R injury and protection. This study investigated the effect of both on myocardial susceptibility to I/R-induced injury and the protective effects of IPreC. Methods and results Atrial myocardium from cardiac surgery patients (n = 300) was assigned to one of three groups: aerobic control, I/R alone, and IPreC. Lactate dehydrogenase leakage, as a marker of cell injury, and cell viability were measured. The basal redox status was determined in samples from 90 patients. The response to I/R varied widely. Myocardium from patients with aortic valve disease was the most susceptible to injury whereas myocardium from dyslipidemia patients was the least susceptible. Tissue from females was better protected than tissue from males. Myocardium from patients with mitral valve disease was the least responsive to IPreC. The basal redox status was altered in the myocardium from patients with mitral and aortic valve disease. Conclusions The response of the myocardium to I/R and IPreC is highly variable and influenced by the underlying cardiac pathology, dyslipidemia, sex, and the basal redox status. These results should be taken into account in the design of future clinical studies on the prevention of I/R injury and protection.This study was supported by the Instituto de Salud Carlos III (FIS) [grant number 12/00119]

Differential Immune Response to Bioprosthetic Heart Valve Tissues in the α1,3Galactosyltransferase-Knockout Mouse Model

Structural valve deterioration (SVD) of bioprosthetic heart valves (BHVs) has great clinical and economic consequences. Notably, immunity against BHVs plays a major role in SVD, especially when implanted in young and middle-aged patients. However, the complex pathogenesis of SVD remains to be fully characterized, and analyses of commercial BHVs in standardized-preclinical settings are needed for further advancement. Here, we studied the immune response to commercial BHV tissue of bovine, porcine, and equine origin after subcutaneous implantation into adult a1,3-galactosyltransferase-knockout (Gal KO) mice. The levels of serum anti-galactose a1,3-galactose (Gal) and -non-Gal IgM and IgG antibodies were determined up to 2 months post-implantation. Based on histological analyses, all BHV tissues studied triggered distinct infiltrating cellular immune responses that related to tissue degeneration. Increased anti-Gal antibody levels were found in serum after ATS 3f and Freedom/Solo implantation but not for Crown or Hancock II grafts. Overall, there were no correlations between cellular-immunity scores and post-implantation antibodies, suggesting these are independent factors differentially affecting the outcome of distinct commercial BHVs. These findings provide further insights into the understanding of SVD immunopathogenesis and highlight the need to evaluate immune responses as a confounding factor

Oxidative Stress in Structural Valve Deterioration : A Longitudinal Clinical Study

The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60-72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months' follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunctio

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Identificación de factores que favorecen la degeneración de las bioprótesis valvulares aórticas

La calcificació és la causa més freqüent de la degeneració estructural de les biopròtesis valvulars aòrtiques. Tot i la seva importància, el mecanisme pel qual el teixit es calcifica segueix sent desconegut. S’han identificat diferents factors que podrien promoure-la, com la presència de fosfolípids de membrana en el teixit protèsic, el tractament químic utilitzat durant la seva fabricació i muntatge, l’estrès mecànic, la resposta de l’hoste contra l’empelt o el dipòsit de cèl·lules i proteïnes diverses. Les teràpies utilitzades des de fa temps per controlar o reduir la calcificació de les biopròtesis han sigut dissenyades seleccionant com a objectius alguns d’aquests factors anteriorment descrits. Aquest treball va demostrà que les biopròtesis Mitroflow LX, sense tractament anticalcificant afegit a la fixació amb glutaraldehid, presenta incidències de degeneració estructural valvular més elevades que la Perimount Magna, sense el mencionat tractament anticalcificant. Tot i que no es va obtenir significació estadística, els pacients d’aquest últim grup van tendir a mostrar millor supervivència després d’homogeneïtzar els grups aplicant una anàlisi per propensió. A més de l’aplicació dels agents anticalcificants, aquest treball va investigar el paper que podrien jugar l’estrès oxidatiu i nitrós sobre el teixit biològic de les vàlvules cardíaques. Entre altres efectes, els radicals lliures produirien una lesió directa del xenoempelt, trencant els enllaços covalents aconseguits amb la fixació amb glutaraldehid. Aquest aspecte li faria perdre la seva estabilitat devant l’estrès mecànic permanent i la seva resistència davant l’acció de les proteïnases circulants. Els resultats van demostrar que els nivells plasmàtics de nitrotirosina i malondialdehid, biomarcadors de l’estrès nitrós i oxidatiu respectivament, en els pacients portadors d’una biopròtesi amb degeneració estructural valvular greu, no van ser significativament diferents dels del grup control, no portadors de biopròtesis. En conseqüència, tampoc es va trobar una activitat antioxidant compensatòria incrementada a través de la quantificació de la “capacitat antioxidant total” en plasma. La conclusió seria que l’estrès oxidatiu probablement tingui un paper efector i/o regulador en l’inici del procés, però que ja es trobi molt atenuat o absent en les fases finals de la malaltia, amb la calcificació establerta i estesa. El fet d’implantar una pròtesi valvular massa petita per a una determinada superfície corporal, pot portar a un grau important de desproporció pròtesi-pacient (PPM), fet que afavoreix la degeneració estructural del teixit amb el pas dels anys. La pròtesi Perceval S no requereix sutura quirúrgica per la seva implantació; la seva vàlvula de pericardi boví va muntada en l’interior d’un suport de nitinol extern de molt poc gruix, fet que permetria implantar aquestes pròtesis en anells nadius més petits dels habituals, oferint tot i això bones àrees efectives de l’orifici valvular (EOA). En aquest context, el nostre estudi va avaluar aquesta pròtesi Perceval amb ecocardiografia Doppler sota condicions d’estrès farmacològic amb dobutamina endovenosa, i es va comparar amb un altre grup que va rebre una pròtesi convencional que sí requeria sutura quirúrgica (Magna-Ease). El grup Perceval va presentar els gradients en repòs lleugerament més elevats que el grup Magna-Ease, EOAs més petites i un percentatge res negligible de PPM greu en el grup. Els seus percentatges d’augment de l’EOA i EOA indexada per la superfície corporal en estrès màxim van ser bons, tot i que sorprenentment iguals que el grup Magna-Ease. Una explicació a aquests resultats inesperats es basaria en la sobreestimació de la mida de les biopròtesis Perceval implantades.La calcificación es la causa más frecuente de la degeneración estructural de las bioprótesis valvulares aórticas. A pesar de su importancia, el mecanismo por el cual el tejido se calcifica sigue siendo desconocido. Se han identificado diferentes factores que podrían promoverla, como la presencia de fosfolípidos en el tejido protésico, el tratamiento químico utilizado durante su fabricación y montaje, el estrés mecánico, la respuesta del huésped al injerto o el depósito de células y proteínas diversas. Las terapias usadas desde hace tiempo para controlar o reducir la calcificación de las bioprótesis han sido diseñadas seleccionando como objetivos a algunos de estos factores anteriormente descritos. Este trabajo demostró que la bioprótesis Mitroflow LX, sin tratamiento anticalcificante añadido a la fijación con gluataraldheído, presenta incidencias de degeneración estructural valvular mucho mayores que la Perimount Magna, con dicho tratamiento anticalcificiante. Aunque no se obtuvo significación estadística, los pacientes de este último grupo tendieron a mostrar mejor supervivencia tras homogeneizar los grupos aplicando un análisis por propensión. Además del uso de los agentes anticalcificantes, este trabajo investigó el papel que podrían jugar el estrés oxidativo y nitroso sobre el tejido biológico de las válvulas cardíacas. Entre otros efectos, los radicales libres producirían una lesión directa del xenoinjerto, rompiendo sus enlaces covalentes conseguidos tras la fijación con glutaraldheído. Este aspecto le haría perder su estabilidad ante el estrés mecánico permanente y su resistencia ante la acción de proteinasas circulantes. Los resultados demostraron que los niveles plasmáticos de nitrotirosina y malondialdheído, biomarcadores del estrés nitroso y oxidativo respectivamente, en los pacientes portadores de una bioprótesis con degeneración estructural grave, no fueron significativamente diferentes del grupo control, no portador de bioprótesis. Por consecuencia, tampoco se halló una actividad antioxidante compensatoria incrementada a través de la cuantificación de la “capacidad antioxidante total” en plasma. La conclusión sería que el estrés oxidativo probablemente tenga un papel efector y/o regulador al inicio del proceso, pero que ya se encuentre muy atenuado o ausente en la fases finales de la enfermedad, con la calcificación establecida y extendida. El implantar una prótesis valvular demasiado pequeña para una determinada superficie corporal, puede conllevar un grado importante de desproporción prótesis-paciente (PPM), hecho que favorece la degeneración estructural del tejido con el paso de los años. La prótesis Perceval S no requiere sutura quirúrgica para su implante; su válvula de pericardio bovino va montada en el interior de un soporte de nitinol externo de muy poco grosor, algo que permitiría implantar estas prótesis en anillos nativos más pequeños de lo habitual, ofreciendo aun así buenas áreas efectivas del orificio valvular (EOA). En este contexto, nuestro estudio evaluó esta prótesis Perceval con ecocardiografía Doppler bajo condiciones de estrés farmacológico con dobutamina endovenosa y se comparó con otro grupo que había recibido una prótesis convencional que sí requería sutura quirúrgica (Magna-Ease). El grupo Perceval presentó los gradientes en reposo algo más elevados que el grupo Magna-Ease, EOAs más pequeñas y un porcentaje nada negligible de PPM severa en el grupo. Sus porcentajes de aumento con estrés máximo del EOA y EOA indexada por la superficie corporal fueron buenos, aunque sorprendentemente iguales que el grupo con la prótesis Magna-Ease. Una explicación a estos resultados inesperados estaría en la sobreestimación del tamaño de las bioprótesis Perceval implantadas.The most common cause of structural valve degeneration is tissue calcification of the aortic bioprostheses. Despite its importance, the mechanisms that would explain this calcification are still not well understood. Some promoting factors have been identified such as the presence of phospholipids in the prosthetic tissue, chemical products used in the manufacturing processes, mechanical stress, host response against the graft, or several proteins and cell debris deposited in the tissue. Therapies used to prevent bioprostheses calcification have been selected according to some of these mentioned promoting factors. This work showed that the Mitroflow LX bioprosthesis, without anti-calcification treatment added after glutaraldehyde fixation process, had a much higher incidence of structural valve degeneration than the Perimount Magna valve, which already had anti-calfication treatment. Furthermore, the latter group showed a not significant trend to have better mid-term survival after adjusting the sample with a propensity score analysis. Beyond the use of anti-calcification treatments, this work investigated the role of both the oxidative and nitrosative stress in the tissue degeneration process of the prosthetic heart valves. Among others, the free radicals may produce a direct lesion to the xenografts, breaking their covalent bonds, and leading to have heart valves much more susceptible to permanent mechanical stress damage and less resistant to the circulating proteinases action. Patients carrying a biosprothesis with structural valve degeneration did not show significantly different plasmatic levels of nitrotirosine and malondialdehyde, biomarkers of nitrosative and oxidative stress respectively, than those of the control group who were not carriers of any bioprosthesis. Consequently, “total antioxidant capacity”was also not increased. The conclusion would be that oxidative stress most likely has an effector and regulatory role at the beginning of the process, but somehow would be attenuated or already absent in the later phases of the disease, once calcification has been established and spread. Severe prosthesis-patient mismatch (PPM) occurs when the inserted heart valve is too small in relation to the patient&#8217;s body surface area, and eventually could lead to structural valve degeneration. The Perceval S valve does not require surgical suture to be implanted, its bovine pericardial valve is mounted inside an external thin stent made of nitinol, something that could facilitate its implantation in the small aortic annulus without loosing effective orifice area (EOA). In this context, our study assessed the Perceval valve with dobutamine stress Doppler echocardiography and was compared to another group that received a conventional bioprosthesis that required surgical suture (Magna-Ease). At rest, the Perceval group had higher mean transprosthetic gradients than the Magna-Ease group, smaller EOAs and a non negligible rate of severe PPM inside the group. Nonetheless, the increasing rates with the maximum stress of the EOA and EOA indexed to body surface area were good, but surprisingly similars to the ones found for the Magna-Ease valve. An explanation to these unexpected outcomes would be the oversizing of the implanted Perceval valves