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

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

Defective dimerization of FoF1-ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging

Aging; Dicarbonyl stress; MitochondriaEnvelliment; Estrès dicarbonílic; MitocondrisEnvejecimiento; Estrés dicarbonílico; MitocondriasAged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1-ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy-dissipating channel involved in cell death. We investigated whether aging alters FoF1-ATP synthase self-assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1-ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1-ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes’ susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1-ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1-ATP synthase glycation in H9c2 myoblasts recapitulated the age-related defective FoF1-ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1-ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.This work was supported by the Instituto de Salud Carlos III of the Spanish Ministry of Health (FIS-PI19-01196) and a grant from the Sociedad Española de Cardiología (SEC/FEC-INV-BAS 217003

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

Ischemic postconditioning of the isolated human myocardium: Role of the applied protocol

Ischemic postconditioning (IPostC), has been proposed as a useful approach to reduce infarct size in all species, but its clinical utility remains unclear. To investigate the role played by the protocol used on the efficacy of IPostC in protecting the diseased human myocardium. Myocardial atrial samples from patients were subjected to a 90 min ischemia/120 min reoxygenation followed by different IPostC protocols to investigate the role of the time of ischemia (30, 60, 90 and 120 s) and the number of cycles (1, 2, 3 and 4) with 60 and 120 s of total ischemic time. Muscles were also subjected to ischemic preconditioning (IPreC). The release of lactate dehydrogenase (LDH) and the measurement of tetrazolium bromide (MTT) were determined. IPostC increased the LDH and decreased the MTT values from those of control, independently of the duration of the conditioning ischemia. LDH and MTT values also worsened by augmenting the number of IPostC cycles whereas they were significantly improved by IPreC. However, analysis of individual results indicated that in approximately 1/3 of the cases IPostC exhibited some degree of protection especially in the presence of increased ischemic injury. The present findings show that IPostC of the human myocardium may be influenced by the protocol used and also by the degree of the preceding ischemic injury. IPostC was beneficial in approximately 1/3 of the cases, however in the remaining cases it increased ischemic damage and, therefore, these results raise a word of caution on its broad clinical use

Ischemic postconditioning of the isolated human myocardium: Role of the applied protocol

Background: Ischemic postconditioning (IPostC), has been proposed as a useful approach to reduce infarct size in all species, but its clinical utility remains unclear. Objective: To investigate the role played by the protocol used on the efficacy of IPostC in protecting the diseased human myocardium. Methods: Myocardial atrial samples from patients were subjected to a 90 min ischemia/120 min reoxygenation followed by different IPostC protocols to investigate the role of the time of ischemia (30, 60, 90 and 120 s) and the number of cycles (1, 2, 3 and 4) with 60 and 120 s of total ischemic time. Muscles were also subjected to ischemic preconditioning (IPreC). The release of lactate dehydrogenase (LDH) and the measurement of tetrazolium bromide (MTT) were determined. Results: IPostC increased the LDH and decreased the MTT values from those of control, independently of the duration of the conditioning ischemia. LDH and MTT values also worsened by augmenting the number of IPostC cycles whereas they were significantly improved by IPreC. However, analysis of individual results indicated that in approximately 1/3 of the cases IPostC exhibited some degree of protection especially in the presence of increased ischemic injury. Conclusions: The present findings show that IPostC of the human myocardium may be influenced by the protocol used and also by the degree of the preceding ischemic injury. IPostC was beneficial in approximately 1/3 of the cases; however in the remaining cases it increased ischemic damage and, therefore, these results raise a word of caution on its broad clinical use