Morusin Alleviates Aortic Valve Calcification by Inhibiting Valve Interstitial Cell Senescence Through Ccnd1/Trim25/Nrf2 Axis.

The senescence of aortic valve interstitial cells (VICs) plays a critical role in the progression of calcific aortic valve disease (CAVD). However, the precise mechanisms underlying the senescence of VICs remain unclear, demanding the identification of a novel target to mitigate this process. Previous studies have highlighted the anti-aging potential of morusin. Thus, this study aimed to explore the therapeutic potential of morusin in CAVD. Cellular experiments reveal that morusin effectively suppresses cellular senescence and cause a shift toward osteogenic differentiation of VICs in vitro. Mechanistically, morusin activate the Nrf2-mediated antiaging signaling pathway by downregulating CCND1 expression and aiding Keap1 degradation through Trim 25. This activation lead to the upregulated expression of antioxidant genes, thus reducing reactive oxygen species production and thereby preventing VIC osteogenic differentiation. In vivo experiments in ApoE-/- mice on a high-fat Western diet demonstrate the positive effect of morusin in mitigating aortic valve calcification. These findings emphasize the antiaging properties of morusin and its potential as a therapeutic agent for CAVD.

Is Time Scheduling Important? An Analysis of Donor Heart Cross-clamp Times During Heart Transplantation.

Background: Outcomes in heart transplantation are affected by a variety of variables and patient factors. However, the impact of circadian rhythms, gene expression, and transcription remain underexplored. We thus evaluated the potential role of donor heart cross-clamp times on short-term and long-term outcomes after heart transplantation. Methods: A total of 31 713 heart transplants were identified from the United Network for Organ Sharing Database. Patients were first stratified on the basis of time of donor procurement: 12 am to 12 pm or 12 pm to 12 am. To evaluate a possible effect of circadian rhythms, donor time was further divided into 5 groups based on preclinical data: 4 am to 8 am; 8 am to 11 am; 11 am to 5 pm; 5 pm to 10 pm; 10 pm to 4 am. Groups were assessed with comparative statistics. Long-term survival was evaluated using Kaplan-Meier methods and a multivariate Cox proportional hazard model. Results: Patients who received hearts recovered between 12 am and 12 pm had significantly higher survival than those who received hearts recovered between 12 pm and 12 am. This survival difference was observed in both unadjusted (P = 0.002) and adjusted analyses (hazard ratio [HR]: 0.93; 95% confidence interval [CI], 0.89-0.97; P < 0.001). On unadjusted analysis, the survival difference among the 5 groups was insignificant (P = 0.07). Following adjustment, the periods of 11 am to 5 pm (HR: 1.09, 95% CI, 1.02-1.17; P = 0.012), 5 pm to 10 pm (HR: 1.11; 95% CI, 1.04-1.19; P = 0.002), and 10 pm to 4 am (HR: 1.07; 95% CI, 1.01-1.15; P = 0.034), were all independently associated with increased long-term mortality. Notably, the time of 8 am to 11 am was not associated with a change in survival (HR: 1.04; 95% CI, 0.96-1.14; P = 0.3). Conclusions: Given the independent association of donor timing and survival after adjustment in a large national cohort, further investigation into the role of donor circadian rhythm and donor procurement time is warranted in preclinical and clinical studies. Understanding the underlying mechanisms of this observation could potentially lead to the development of effective treatments and donor procurement processes that prepare the organs for transplantation in a better condition.

Directed Differentiation of Human Induced Pluripotent Stem Cells to Heart Valve Cells.

BACKGROUND: A main obstacle in current valvular heart disease research is the lack of high-quality homogeneous functional heart valve cells. Human induced pluripotent stem cells (hiPSCs)-derived heart valve cells may help with this dilemma. However, there are no well-established protocols to induce hiPSCs to differentiate into functional heart valve cells, and the networks that mediate the differentiation have not been fully elucidated. METHODS: To generate heart valve cells from hiPSCs, we sequentially activated the Wnt, BMP4, VEGF (vascular endothelial growth factor), and NFATc1 signaling pathways using CHIR-99021, BMP4, VEGF-165, and forskolin, respectively. The transcriptional and functional similarity of hiPSC-derived heart valve cells compared with primary heart valve cells were characterized. Longitudinal single-cell RNA sequencing was used to uncover the trajectory, switch genes, pathways, and transcription factors of the differentiation. RESULTS: An efficient protocol was developed to induce hiPSCs to differentiate into functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells. After 6-day differentiation and CD144 magnetic bead sorting, ≈70% CD144+ cells and 30% CD144- cells were obtained. On the basis of single-cell RNA sequencing data, the CD144+ cells and CD144- cells were found to be highly similar to primary heart valve endothelial cells and primary heart valve interstitial cells in gene expression profile. Furthermore, CD144+ cells had the typical function of primary heart valve endothelial cells, including tube formation, uptake of low-density lipoprotein, generation of endothelial nitric oxide synthase, and response to shear stress. Meanwhile, CD144- cells could secret collagen and matrix metalloproteinases, and differentiate into osteogenic or adipogenic lineages like primary heart valve interstitial cells. Therefore, we identified CD144+ cells and CD144- cells as hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells, respectively. Using single-cell RNA sequencing analysis, we demonstrated that the trajectory of heart valve cell differentiation was consistent with embryonic valve development. We identified the main switch genes (NOTCH1, HEY1, and MEF2C), signaling pathways (TGF-ß, Wnt, and NOTCH), and transcription factors (MSX1, SP5, and MECOM) that mediated the differentiation. Finally, we found that hiPSC-derived valve interstitial-like cells might derive from hiPSC-derived valve endothelial-like cells undergoing endocardial-mesenchymal transition. CONCLUSIONS: In summary, this is the first study to report an efficient strategy to generate functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells from hiPSCs, as well as to elucidate the differentiation trajectory and transcriptional dynamics of hiPSCs differentiated into heart valve cells.

BATF and BATF3 deficiency alters CD8+ effector/exhausted T cells balance in skin transplantation.

BACKGROUND: It is well-established that CD8+ T-cells play a critical role in graft rejection. The basic leucine zipper ATF-like transcription factor (BATF) and BATF3 are transcriptional factors expressed in T lymphocytes. Herein, we investigated the functions of BATF and BATF3 in the differentiation and exhaustion of CD8+ T cells following alloantigen activation. METHODS: Wild-type CD8+ T cells, BATF-deficient (Batf-/-) CD8+ T cells, and CD8+ T cells deficient in both BATF and BATF3 (Batf-/-Batf3-/-) were transferred to B6.Rag1-/- mice, which received skin allografts from BALB/c mice. Flow cytometry was conducted to investigate the number of CD8+ T cells and the percentage of effector subsets. RESULTS: BATF expression positively correlated with effector CD8+ T cell differentiation. BATF and BATF3 deficiency promoted skin allograft long-term survival and attenuated the CD8+ T cell allo-response and cytokine secretion. Finally, BATF and BATF3 deficiency prompted the generation of exhausted CD8+ T cells. CONCLUSIONS: Overall, our findings provide preliminary evidence that both BATF and BATF3 deficiency influences the differentiation of effector CD8+ T cells and mediates the exhaustion of CD8+ T cells, prolonging transplant survival. Targeting BATF and BATF3 to inhibit CD8+ T cell function has huge prospects for application as a therapeutic approach to prevent transplant rejection.

Donor circadian clock influences the long-term survival of heart transplantation by immunoregulation.

AIMS: Circadian clocks play important role in immunoregulation. We aimed to investigate cardiac circadian clock specific pathways and compare cardiac grafts procured at different timing on survival after transplantation to explore novel criteria for donor selection. METHODS AND RESULTS: In primate heart, phase set enrichment analysis (PSEA) showed rhythmic transcripts were enriched in antigen processing and presentation during activation of circadian rhythm. Digital sorting of immune cell composition and single-sample gene set enrichment analysis (ssGSEA) in unused donor transcriptomes showed the pathway, positive regulation of circadian rhythm significantly correlates with allograft rejection and antigen presentation pathways as well as with increased compositions of matured dendritic cell, CD4+ T cell, and naive B cell. Single-centre retrospective cohort of 390 adult heart transplants between 1 January 2015 and 31 December 2020 was used to generate a propensity score matching (PSM) cohort. Survival curve differed significantly showing inferior long-term survival when donor hearts were procured at activation group (12 pm to 12 am) compared to repression group (12 am to 12 pm) (6-year survival: 64.2% vs. 75.8%, P = 0.0065). Activation group was also associated with significantly higher rates of in-hospital death, cardiopulmonary resuscitation, and usage of mechanical circulatory support after heart transplantation compared to repression group. Furthermore, tendency for post-transplant free of rejection rates was higher in repression group compared to activation group (acute rejection, Gehan-Breslow P = 0.11 and 0.04; chronic rejection, Log rank P = 0.077 and 0.15, in full and PSM cohorts, respectively). Adjusted Cox regression analysis showed that activation group was associated with 2.20 times increased hazard of death (hazard ratio: 2.20; 95% confidence interval: 1.23-3.95; P = 0.008) compared to repression group. CONCLUSIONS: Circadian immunity may represent donor-related risk factors for cardiac allograft rejection through activating genes related to antigen presentation pathway and immune cells oscillation at specific time of day. Molecular circadian clock should be considered during retrieval of cardiac allografts in order to maximize graft durability.

Platelet membrane-coated alterbrassicene A nanoparticle inhibits calcification of the aortic valve by suppressing phosphorylation P65 NF-κB.

Rationale: Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and morbidity with increasing prevalence and incidence. The pathobiology of CAVD involves valvular fibrocalcification, and osteogenic and fibrogenic activities are elevated in aortic valve interstitial cells (VICs) from diseased valves. It has been demonstrated that activated NF-κB pathway was present in the early stage of CAVD process. There is currently no effective clinical drugs targeting NF-κB pathway for CAVD treatment. Therefore, it is of great clinical significance to seek effective treatments for valve calcification. Methods: In this study, we established immortal human valve interstitial cells (im-hVICs) with pGMLV-SV40T-puro lentivirus. Alizarin red staining and western blotting were performed to evaluate the calcification of immortal VICs supplemented with different compounds. The natural fusicoccane diterpenoid alterbrassicene A (ABA) was found to have potential therapeutic functions. Ribonucleic acid sequencing was used to identify the potential target of ABA. Platelet membrane-coated nanoparticle of ABA (PNP-ABA) was fabricated and the IBIDI pump was used to evaluate the adhesion ability of PNP-ABA. Murine wire-induced aortic valve stenosis model was conducted for in vivo study of PNP-ABA. Results: The natural fusicoccane diterpenoid ABA was found to significantly reduce the calcification of human VICs during osteogenic induction via inhibiting the phosphorylation P65. Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein-2 (BMP2) were down regulated with the treatment of ABA in human VICs. Additionally, molecular docking results revealed that ABA bound to RelA (P65) protein. Phosphorylation of P65 (Ser536) was alleviated by ABA treatment, as well as the nuclear translocation of P65 during osteogenic induction in human VICs. Alizarin red staining showed that ABA inhibited osteogenic differentiation of VICs in a dose-dependent manner. PNP-ABA attenuated aortic valve calcification in murine wire-induced aortic valve stenosis model in vivo. Conclusions: The establishment of im-hVICs provides a convenient cell line for the study of CAVD. Moreover, our current research highlights a novel natural compound, ABA, as a promising candidate to prevent the progression of CAVD.

Mechanisms and Drug Therapies of Bioprosthetic Heart Valve Calcification.

Valve replacement is the main therapy for valvular heart disease, in which a diseased valve is replaced by mechanical heart valve (MHV) or bioprosthetic heart valve (BHV). Since the 2000s, BHV surpassed MHV as the leading option of prosthetic valve substitute because of its excellent hemocompatible and hemodynamic properties. However, BHV is apt to structural valve degeneration (SVD), resulting in limited durability. Calcification is the most frequent presentation and the core pathophysiological process of SVD. Understanding the basic mechanisms of BHV calcification is an essential prerequisite to address the limited-durability issues. In this narrative review, we provide a comprehensive summary about the mechanisms of BHV calcification on 1) composition and site of calcifications; 2) material-associated mechanisms; 3) host-associated mechanisms, including immune response and foreign body reaction, oxidative stress, metabolic disorder, and thrombosis. Strategies that target these mechanisms may be explored for novel drug therapy to prevent or delay BHV calcification.

Dose approach matter? A meta-analysis of outcomes following transfemoral versus transapical transcatheter aortic valve replacement.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) has gained increasing acceptance for patients with aortic disease. Both transfemoral (TF-TAVR) and transapical (TA-TAVR) approach were widely adopted while their performances are limited to a few studies with controversial results. This meta-analysis aimed to compare the mortality and morbidity of complications between TF- versus TA-TAVR based on the latest data. METHODS: Electronic databases were searched until April 2021. RCTs and observational studies comparing the outcomes between TF-TAVR versus TA-TAVR patients were included. Heterogeneity assumption was assessed by an I2 test. The pooled odds ratios(OR) or mean differences with corresponding 95% confidence intervals (CI) were used to evaluate the difference for each end point using a fixed-effect model or random-effect model based on I2 test. RESULTS: The meta-analysis included 1 RCT and 20 observational studies, enrolling 19,520 patients (TF-TAVR, n = 11,986 and TA-TAVR, n = 7,534). Compared with TA-TAVR, TF-TAVR patients showed significantly lower rate of postoperative in-hospital death (OR = 0.67, 95% CI 0.59-0.77, P < 0.001) and 1-year death (OR = 0.53, 95% CI 0.41-0.69, P < 0.001). Incidence of major bleeding and acute kidney injury were lower and length of hospital stay was shorter, whereas those of permanent pacemaker and major vascular complication were higher in TF-TAVR patients. There were no significant differences between TF-TAVR versus TA-TAVR for stroke and mid-term mortality. CONCLUSIONS: There were fewer early deaths in patients with transfemoral approach, whereas the number of mid-term deaths and stroke was not significantly different between two approaches. TF-TAVR was associated with lower risk of bleeding, acute kidney injury as well as shorter in-hospital stay, but higher incidence of vascular complication and permanent pacemaker implantation.

Predictors of Prosthetic Valve Endocarditis following Transcatheter Aortic Valve Replacement: A Meta-Analysis.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) has gained increasing acceptance for patients with aortic disease. A rare but fatal complication prosthetic valve endocarditis (PVE) could greatly influence the clinical outcomes of TAVR. This meta-analysis aims to pin down the predictors of PVE in TAVR patients. METHODS: We performed a systematic search for studies that reported the incidence and risk factors of PVE after TAVR. Data on studies, patients, baseline characteristics, and procedural characteristics were abstracted. Crude risk ratios (RRs) and 95% confidence intervals for each predictor were calculated by the use of random-effects models. Heterogeneity assumption was assessed by an I2 test. RESULTS: We obtained data from 8 studies that included 68,805 TAVR patients, of whom 1,256 (1.83%) were diagnosed with PVE after TAVR. 280 patients died within the 30-days of PVE diagnosis and the pooled in-hospital mortality was 22.3%. The summary estimates indicated an increased risk of PVE after TAVR for males (RR 1.53, P = .0001); for patients with orotracheal intubation (RR 1.65, P = .01), new pacemaker implantation (RR 1.46, P = .003), and residual aortic regurgitation (≥2 grade) (RR 1.62, P = .05); while older age (RR 0.97, P = .0007) and implantation of a self-expandable valve (RR 0.74, P = .02) were associated with a lower risk of PVE after TAVR. CONCLUSION: Clinical characteristics and peri- procedure factors including age, male sex, valve type, orotracheal intubation, pacemaker implantation, and residual regurgitation were proven to be associated with the occurrence of PVE-TAVR. Clinicians should pay particular attention to PVE when treating TAVR patients with these predictors.

Bioprosthetic vs mechanical mitral valve replacement for infective endocarditis in patients aged 50 to 69 years.

BACKGROUND: The optimal choice of the valve prosthesis in mitral valve replacement (MVR) for infective endocarditis (IE) is controversial and challenging, particularly for younger patients. HYPOTHESIS: The postoperative outcomes of mechanical and biological MVR in IE patients aged 50 to 69 years are different. METHODS: All IE patients aged 50 to 69 years with primary MVR in Hubei province hospitals from 2002 to 2018 were retrospectively reviewed. The median duration of follow-up was 8.7 years (IQR, 6.8-10.9 years). Propensity score matching (1:3 ratio) was used to yield 492 patients with comparable baseline features between bioprostheses and mechanical prosthetic valve groups. Outcomes were postoperative mid- to long- term survival, mitral valve reoperation, prosthetic valve endocarditis (PVE), stroke, and major bleeding events. RESULTS: Fifteen-year survival after MVR was 80.6% in the mechanical valve group and 69.3% in the bioprostheses group (HR 0.545, P = .040). The cumulative incidence of mitral valve reoperation was 8.8% with mechanical valves and 21.4% with bioprostheses (HR 0.260, P = .002). The cumulative incidence of PVE was 5.6% with mechanical valves and 7.2% with bioprostheses (HR 0.629, P = .435). The cumulative incidence of stroke was 12.9% with mechanical valves and 10.5% with bioprostheses (HR 1.217, P = .647). The cumulative incidence of major bleeding was 12.0% with mechanical valves and 6.75% with bioprostheses (HR 1.579, P = .268). CONCLUSIONS: Mechanical valve prostheses were associated with better survival, lower rates of reoperation compared with bioprostheses within 15 years after MVR in IE patients aged 50 to 69. These findings suggest mechanical valve prostheses may be a more reasonable alternative to bioprostheses in this patient group.