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 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.

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 dysfunction.

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

Aged 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.

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

Bioprosthetic 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.

Characterization of putative regulatory isoforms of porcine tumor necrosis factor receptor 2 in endothelial cells.

Tumor necrosis factor α (TNFα) and its receptors contribute to rejection of transplanted cells and organs. To elucidate how TNFα affects xenograft rejection, we previously cloned the cDNA of pig TNF-receptor 2 (pTNFR2) and found four isoforms: one comprising the full receptor with four cysteine-rich domains (CRD), a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another lacking exon 4 (pTNFR2ΔE4) displaying only 3 CRD and poor ligand binding, and the smallest one generated by the two alternative splicings. All isoforms contained the pre-ligand assembly domain (PLAD) responsible for receptor trimerization. We now investigated their roles by structural, expression, and subcellular localization studies. Structural in silico analyses identified four amino acids potentially involved in TNFα binding and lacking in pTNFR2ΔE4. Quantitative RT-PCR determined regulated expression affecting the two pTNFR2 alternative splicings in cytokine-stimulated porcine aortic endothelial cells (PAEC). Particularly, human IL-1α and TNFα produced a strong mRNA upregulation of all isoforms, being the full receptor the predominant one. However, expression of pTNFR2 on PAEC did not correlate with mRNA and decreased after 24-hour exposure to IL-1α or TNFα. Notably, confocal microscopy confirmed the presence of pTNFR2 inside and on the plasma membrane, whereas pTNFR2ΔE4 located only intracellularly. Most interestingly, FRET analyses showed that membrane-bound isoforms pTNFR2 and pTNFR2ΔE4 colocalized intracellularly and associated through the PLAD. Our data show that pTNFR2ΔE4 bind and may retain the full receptor intracellularly. This mechanism has not been described in other species and represents a particularity that may affect the pathophysiology of pig xenografts.

Cell-Based Assays for Modeling Xenogeneic Immune Responses.

Research in xenotransplantation implies a high experimental complexity comprising molecular, cellular, and in vivo studies to investigate the mechanisms of xenograft immune rejection and functional failure, as well as the strategies to counteract them. After major advances associated with the identification of the carbohydrate xenoantigens and their elimination through genomic edition of the source pigs, the study of the cellular immune response against the xenograft is gaining particular attention. Xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells are relevant to address this hurdle. Thus, we describe here coculture, co-stimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms of xenograft rejection. These techniques allow elucidating the key pathways that take place during the xenogeneic immune response in a simplified setting. Treatment with either pro-inflammatory or anti-inflammatory cytokines can be used for studying the regulation of adhesion, co-stimulatory molecules, and receptors involved in triggering the immune response under various conditions. Furthermore, these assays can be used for the follow-up of the immune response of in vivo studies as well as for the development of tolerogenic approaches that promote xenograft survival.

Determination of Redox Status in Serum.

Free radicals of oxidative and nitrosative stress can trigger both pro-inflammatory and anti-inflammatory responses. In the transplant setting, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced at the rejection site by different cell types including endothelial cells and macrophages. In particular, production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) seems to play an important role in promoting inflammation after exposure to inflammatory stimuli. In xenotransplantation, NO produced by iNOS upregulate multiple vasoactive substances, cytokines, chemokines, and growth factors, whereas production of NO by endothelial nitric oxide synthase (eNOS) could confer a protective effect to the graft. Accordingly, further research is needed to better understand the associated mechanisms in order to enhance protection and prevent tissue damage. Here, we describe simple methods to determine the redox state in serum that could be applied to animal models such as for xenotransplantation studies, as well as to clinical samples. Notably, caution should be taken when interpreting results of ROS and RNS measurements due to this dual role of free radicals in protecting and injuring the graft.

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

BACKGROUND: 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.

Effect of bioprostheses anti-calcification treatment: comparative follow-up between Mitroflow LX and Magna pericardial xenografts using a propensity score-weighted analysis.

Objectives: The efficacy of anti-calcification treatment of bioprosthetic heart valves remains unclear. The aim of this study was to compare the clinical outcomes between Mitroflow LX valve, without anti-calcification treatment, and the Carpentier-Edwards Perimount Magna (P-Magna), with anti-calcification treatment. Methods: Between 2005 and 2012, 625 consecutive patients underwent aortic valve replacement either with a Mitroflow LX ( n = 329) or a P-Magna ( n = 296). Variables regarding patient-related risk factors and operative data were accounted for an inverse probability of treatment weighting analysis. Then, adjusted survival outcomes and the rate of structural valve disease (SVD) were assessed for each group. Results: Mean follow-up times were 4.1 ± 2.29 years and 3.9 ± 2.63 years, respectively ( P = 0.34). Adjusted overall survival rate was higher in the P-Magna group than in the Mitroflow LX group at 8 years (69.1% vs 51.9%, respectively) [HR = 1.44, 95% CI: 1.01 to 2.06; P = 0.0467]. Similarly, the 8-year cardiac-related survival rate was also higher in the P-Magna group [HR = 1.99, 95% CI: 1.19 to 3.32; P = 0.0083]. One patient (0.8%) with P-Magna and 23 patients (18.5%) with Mitroflow LX group developed SVD (0.24% per patient-year vs 4.5% per patient-year, respectively; P < 0.001). At 5 and 8 years, valve-related survival rates did not differ significantly between both groups [HR = 1.67, 95% CI: 0.95 to 2.95; P = 0.075]. Conclusions: The P-Magna prosthesis showed significantly better overall and cardiac-related survival than the Mitroflow LX. The higher early SVD and reoperation rates seen with the Mitroflow LX prosthesis did not impact negatively on valve-related survival.

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.

De-alcoholised white and red wines decrease inflammatory markers and NF-κB in atheroma plaques in apoE-deficient mice.

AIMS OF THE STUDY: Wine polyphenols attenuate the development of atherosclerosis, which involves an inflammatory process. We studied the beneficial effect of de-alcoholised white and red wines (DWW and DRW, respectively) on the development of atheroma plaques and on the expression of biomarkers. METHODS: We administered control or de-alcoholised wine-rich diets to apoE-deficient mice for 12 or 20 weeks. We then used optical microscopy or immunofluorescence to examine atherosclerotic lesion development in the thoracic aorta and aortic root and assessed the presence of cytokines and adhesion molecules by qPCR and immunofluorescence in total aorta and aortic root, respectively. RESULTS: Atherosclerotic lesions in thoracic aorta were significantly decreased in mice supplemented with DWW (30 %) and DRW (62 %) for 20 weeks. In addition, the expressions of interferon-γ, interleukin-1ß, the monocyte chemoattractant protein-1 and CD68 were reduced by DRW. The adhesion molecule P-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 were decreased by 52, 76 and 45 %, respectively, in mice fed DRW for 12 weeks, whereas DWW reduced these parameters in a minor extent. The NF-κB expression in total aorta was significantly decreased in the mice treated with de-alcoholised wines for 12 weeks. CONCLUSIONS: DRW is shown to be more effective than DWW on cytokines and adhesion molecule expression, in the early stages of the inflammatory events associated with atherosclerosis development, probably due to the high phenolic content of red wine. Downregulation of NF-κB expression may be involved in the mechanism by which de-alcoholised wines modulate atherosclerosis.

Tumor cells induce COX-2 and mPGES-1 expression in microvascular endothelial cells mainly by means of IL-1 receptor activation.

Prostaglandin (PG) E(2) plays a key role in immune response, tumor progression and metastasis. We previously showed that macrovessel-derived endothelial cells do not produce PGE(2) enzymatically because they do not express the inducible microsomal PGE-synthase-1 (mPGES-1). Nevertheless, differences between macro- and micro-vessel-derived endothelial cells regarding arachidonic acid (AAc) metabolism profile have been reported. The present work was conducted to evaluate the expression of PGE(2)-pathway-related enzymes in human microvascular endothelial cells (HMVEC) in culture and to test the hypothesis that the tumor cell-HMVEC cross talk could increase mPGES-1 expression in HMVEC. We treated HMVEC in culture with human recombinant IL-1ß. IL-1ß induced PGE(2) release and COX-2 and mPGES-1 expression in terms of mRNA and protein, determined by real-time PCR and immunoblotting, respectively. HMVEC constitutively expressed mPGES-2 and cytosolic PGES (cPGES) and the IL-1ß treatment did not modify their expression. PGE(2) synthesized by HMVEC from exogenous AAc was linked to mPGES-1 expression. Immunohistochemistry analysis confirmed mPGES-1 expression in microvessels in vivo. COX-2 and mPGES-1 were also induced in HMVEC by the conditioned medium from two squamous head and neck carcinoma cell lines. Conditioned medium from tumor cell cultures contained several cytokines including the IL-1ß and IL-1α. Tumor cell-induced COX-2 and mPGES-1 in HMVEC was strongly inhibited by the IL-1-receptor antagonist, indicating the important implication of IL-1 in this effect. HMVEC could therefore contribute directly to PGE(2) formed in the tumor. Our findings support the concept that mPGES-1 could be a target for therapeutic intervention in patients with cancer.

A fish-oil-rich diet reduces vascular oxidative stress in apoE(-/-) mice.

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE(-/-) mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O(2)(.-)) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22(phox) expression and O(2)(.-)production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.

Atherosclerosis prevention by a fish oil-rich diet in apoE(-/-) mice is associated with a reduction of endothelial adhesion molecules.

Dietary intake of long-chain n-3 polyunsaturated fatty acids (PUFA) reduces the risk for atherosclerosis. Here we examine the effect of a fish oil (FO)-rich diet on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. Mice were fed semi-purified diets containing 5% corn oil (CO), rich in n-6 PUFA or menhaden oil as FO, rich in long-chain n-3 PUFA and 0.15% cholesterol after reaching 4 weeks of age, and they were killed when they were 4 weeks, 12 weeks, 18 weeks or 24 weeks old. Oxidative stress in plasma and aortic tissue was not increased in mice fed the FO-rich diet, despite its high peroxidizability index. A reduction of stenosis and intrusion at the aortic root, a decrease in the surface area of atherosclerotic lesions at the aorta and a decrease in P-selectin, vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression were observed in FO-fed mice compared to CO-fed mice. It seems likely that the reduced expression of VCAM-1 and ICAM-1 could be transcriptionally regulated by nuclear factor-kappaB in the aortic root. The protective effect of FO against atherosclerosis was more evident at early ages. In conclusion, FO reduces adhesion molecule expression in lesions in apoE(-/-) mice. Because these molecules are involved in lesion progression the effect of FO may explain the observed decrease in atherogenesis.

Long-chain n-3 polyunsaturated fatty acid from fish oil modulates aortic nitric oxide and tocopherol status in the rat.

In spite of their high oxidisability, long-chain n-3 PUFA protect against CVD. Dietary fatty acids modulate the fatty acid composition of lipoproteins involved in atherosclerosis. We thought that if long-chain n-3 PUFA were able to increase NO production by the aorta, then by its antioxidant activity the NO will prevent lipid peroxidation. However, the beneficial effect of NO in vivo on VLDL + LDL oxidation would only be possible if NO could diffuse to their lipidic core. Rats were fed maize oil- or fish oil as menhaden oil- (MO) rich diets for 8 weeks, to study the effects of MOon aortic NO production, NO diffusion into VLDL + LDL, the extent of oxidation in native VLDL + LDL and their oxidisability ex vivo. Aortic NO production and its alpha-tocopherol content were increased and n-3 PUFA were incorporated into the VLDL + LDL. In spite of the higher peroxidisability and the low alpha-tocopherol in native VLDL + LDL from rats fed MO, native VLDL + LDL from the two groups shared similar electrophoretic patterns, conjugated dienes, thiobarbituric acid-reactive substances, total antioxidant capacity, and NO diffusibility on VLDL + LDL, indicative of an in vivo protection against oxidation. However, these results do not correlate with the ex vivo oxidisability of VLDL+ LDL, as NO is lacking. Thus, the in vivo beneficial effects can be explained by increased a-tocopherol in aorta and by a compensatory effect of NO onVLDL + LDL against the low alpha-tocopherol levels, which may contribute to the anti-atherogenic properties of fish oil.

Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet.

A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the.NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular.NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, l-arginine, and l-citrulline were also evaluated in homogenates. Superoxide anion (O(2)(-).) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%);.NO production increased by 90%, cGMP levels increased by 100%, and l-arginine decreased by 30%. No change in aortic O(2)(-). production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.