ACLY as a modulator of liver cell functions and its role in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND: Non-alcoholic Fatty Liver Disease (NAFLD), now better known as Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and its progression to Nonalcoholic Steatohepatitis (NASH), more recently referred to as Metabolic (Dysfunction)-Associated Steatohepatitis (MASH) are the most common causes of liver failure and chronic liver damage. The new names emphasize the metabolic involvement both in relation to liver function and pathological features with extrahepatic manifestations. This study aims to explore the role of the immunometabolic enzyme ATP citrate lyase (ACLY), with a critical function in lipogenesis, carbohydrate metabolism, gene expression and inflammation. METHODS: ACLY function was investigated in TNFα-triggered human hepatocytes and in PBMC-derived macrophages from MASH patients. Evaluation of expression levels was carried out by western blotting and/or RT-qPCR. In the presence or absence of ACLY inhibitors, ROS, lipid peroxidation and GSSG oxidative stress biomarkers were quantified. Chromatin immunoprecipitation (ChIP), transient transfections, immunocytochemistry, histone acetylation quantitation were used to investigate ACLY function in gene expression reprogramming. IL-6 and IL-1ß were quantified by Lumit immunoassays. RESULTS: Mechanistically, ACLY inhibition reverted lipid accumulation and oxidative damage while reduced secretion of inflammatory cytokines in TNFα-triggered human hepatocytes. These effects impacted not only on lipid metabolism but also on other crucial features of liver function such as redox status and production of inflammatory mediators. Moreover, ACLY mRNA levels together with those of malic enzyme 1 (ME1) increased in human PBMC-derived macrophages from MASH patients when compared to age-matched healthy controls. Remarkably, a combination of hydroxycitrate (HCA), the natural ACLY inhibitor, with red wine powder (RWP) significantly lowered ACLY and ME1 mRNA amount as well as IL-6 and IL-1ß production in macrophages from subjects with MASH. CONCLUSION: Collectively, our findings for the first time highlight a broad spectrum of ACLY functions in liver as well as in the pathogenesis of MASH and its diagnostic and therapeutic potential value.

The Intrinsic Virtues of EGCG, an Extremely Good Cell Guardian, on Prevention and Treatment of Diabesity Complications.

The pandemic proportion of diabesity-a combination of obesity and diabetes-sets a worldwide health issue. Experimental and clinical studies have progressively reinforced the pioneering epidemiological observation of an inverse relationship between consumption of polyphenol-rich nutraceutical agents and mortality from cardiovascular and metabolic diseases. With chemical identification of epigallocatechin-3-gallate (EGCG) as the most abundant catechin of green tea, a number of cellular and molecular mechanisms underlying the activities of this unique catechin have been proposed. Favorable effects of EGCG have been initially attributed to its scavenging effects on free radicals, inhibition of ROS-generating mechanisms and upregulation of antioxidant enzymes. Biologic actions of EGCG are concentration-dependent and under certain conditions EGCG may exert pro-oxidant activities, including generation of free radicals. The discovery of 67-kDa laminin as potential EGCG membrane target has broaden the likelihood that EGCG may function not only because of its highly reactive nature, but also via receptor-mediated activation of multiple signaling pathways involved in cell proliferation, angiogenesis and apoptosis. Finally, by acting as epigenetic modulator of DNA methylation and chromatin remodeling, EGCG may alter gene expression and modify miRNA activities. Despite unceasing research providing detailed insights, ECGC composite activities are still not completely understood. This review summarizes the most recent evidence on molecular mechanisms by which EGCG may activate signal transduction pathways, regulate transcription factors or promote epigenetic changes that may contribute to prevent pathologic processes involved in diabesity and its cardiovascular complications.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

Protein Phosphatase 1 Beta is Modulated by Chronic Hypoxia and Involved in the Angiogenic Endothelial Cell Migration.

BACKGROUND/AIM: Endothelial cell migration is required for physiological angiogenesis, but also contributes to various pathological conditions, including tumour vascularization. The mRNA expression of PP1cß, the beta isoform of the catalytic PP1 subunit, was shown to be upregulated in chronic hypoxia. Since hypoxia is a major regulator of angiogenesis, the potential role of PP1cß in angiogenesis was investigated. METHODS: We examined PP1cß protein level in pediatric heart following chronic hypoxia and found PP1cß upregulation in cyanotic compared with acyanotic myocardium. By treating HUVEC cells with hypoxia mimicking agent, PP1cß protein level increased with maximum at 8 hours. The effect of PP1cß pharmacological inhibition, knockdown and overexpression, on endothelial cell migration and morphogenesis, was examined using in vitro wound healing scratch assay and endothelial tube formation assay. The PP1cß knockdown effects on F-actin reorganization (phalloidin staining), focal adhesion formation (vinculin) and focal adhesion kinases (FAK) activation, were evaluated by immunocytochemical staining and immunoblotting with specific antibodies. RESULTS: PP1cß knockdown significantly reduces endothelial cell migration, but does not have any significant effect on endothelial tube formation. Endothelial cell migration in the knockdown group is restored to the control level upon consecutive transfection with PP1cß cDNA. PP1cß overexpression does not significantly affect endothelial cell migration. Furthermore, PP1cß knockdown induces profound cytoskeletal reorganization, loss of focal adhesion sites and impairment of focal adhesion kinases (FAK) activation. CONCLUSIONS: PP1cß is regulator of endothelial cell migration, which is critical in the angiogenic process. PP1cß inhibition reduces endothelial cell migration through focal adhesion turnover and actin polymerization pathways.

Protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber.

Surgical treatment of pediatric congenital heart disease with tissue grafts is a lifesaving intervention. Decellularization to reduce immunogenicity of tissue grafts is an increasingly popular alternative to glutaraldehyde fixation. Here, we present a protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber. We describe steps for 3D printing the flow rig, preparing porcine tissue, and using the flow rig to utilize shear forces for decellularization. We then detail procedures for characterizing the acellular scaffold. For complete details on the use and execution of this protocol, please refer to Vafaee et al.1.

Growth capacity of a Wharton's Jelly derived mesenchymal stromal cells tissue engineered vascular graft used for main pulmonary artery reconstruction in piglets.

Background: Surgical treatment of congenital heart defects affecting the right ventricular outflow tract (RVOT) often requires complex reconstruction and multiple reoperations due to structural degeneration and lack of growth of currently available materials. Hence, alternative approaches for RVOT reconstruction, which meet the requirements of biocompatibility and long-term durability of an ideal scaffold, are needed. Through this full scale pre-clinical study, we demonstrated the growth capacity of a Wharton's Jelly derived mesenchymal stromal cells (WJ-MSC) tissue engineered vascular graft used in reconstructing the main pulmonary artery in piglets, providing proof of biocompatibility and efficacy. Methods: Sixteen four-week-old Landrace pigs were randomized to undergo supravalvar Main Pulmonary Artery (MPA) replacement with either unseeded or WJ-MSCs-seeded Small Intestinal Submucosa-derived grafts. Animals were followed up for 6 months by clinical examinations and cardiac imaging. At termination, sections of MPAs were assessed by macroscopic inspection, histology and fluorescent immunohistochemistry. Results: Data collected at 6 months follow up showed no sign of graft thrombosis or calcification. The explanted main pulmonary arteries demonstrated a significantly higher degree of cellular organization and elastin content in the WJ-MSCs seeded grafts compared to the acellular counterparts. Transthoracic echocardiography and cardiovascular magnetic resonance confirmed the superior growth and remodelling of the WJ-MSCs seeded conduit compared to the unseeded. Conclusion: Our findings indicate that the addition of WJ-MSCs to the acellular scaffold can upgrade the material, converting it into a biologically active tissue, with the potential to grow, repair and remodel the RVOT.

New Insights into NF-κB Signaling in Innate Immunity: Focus on Immunometabolic Crosstalks.

The nuclear factor kappa B (NF-κB) is a family of transcription factors that, beyond their numberless functions in various cell processes, play a pivotal role in regulating immune cell activation. Two main pathways-canonical and non-canonical-are responsible for NF-κB activation and heterodimer translocation into the nucleus. A complex crosstalk between NF-κB signaling and metabolism is emerging in innate immunity. Metabolic enzymes and metabolites regulate NF-κB activity in many cases through post-translational modifications such as acetylation and phosphorylation. On the other hand, NF-κB affects immunometabolic pathways, including the citrate pathway, thereby building an intricate network. In this review, the emerging findings about NF-κB function in innate immunity and the interplay between NF-κB and immunometabolism have been discussed. These outcomes allow for a deeper comprehension of the molecular mechanisms underlying NF-κB function in innate immune cells. Moreover, the new insights are important in order to perceive NF-κB signaling as a potential therapeutic target for inflammatory/immune chronic diseases.

Graft rejection in paediatric congenital heart disease.

Congenital heart disease (CHD) affects around 1.35 million neonates worldwide per annum, and surgical repair is necessary in approximately 25% of cases. Xenografts, usually of bovine or porcine origin, are often used for the surgical reconstruction. These xenografts elicit an immune response due to significant immunological incompatibilities between host and donor. Current techniques to dampen the initial hyperacute rejection response involve aldehyde fixation to crosslink xenoantigens, such as galactose-α1,3-galactose and N-glycolylneuraminic acid. While this temporarily masks the epitopes, aldehyde fixation is a suboptimal solution, degrading over time, resulting in cytotoxicity and rejection. The immune response to foreign tissue eventually leads to chronic inflammation and subsequent graft failure, necessitating reintervention to replace the defective bioprosthetic. Decellularisation to remove immunoincompatible material has been suggested as an alternative to fixation and may prove a superior solution. However, incomplete decellularisation poses a significant challenge, causing a substantial immune rejection response and subsequent graft rejection. This review discusses commercially available grafts used in surgical paediatric CHD intervention, looking specifically at bovine jugular vein conduits as a substitute to cryopreserved homografts, as well as decellularised alternatives to the aldehyde-fixed graft. Mechanisms of biological prosthesis rejection are explored, including the signalling cascades of the innate and adaptive immune response. Lastly, emerging strategies of intervention are examined, including the use of tissue from genetically modified pigs, enhanced crosslinking and decellularisation techniques, and augmentation of grafts through in vitro recellularisation or functionalisation with human surface proteins.

Corrigendum: Accelerated cardiac aging in patients with congenital heart disease.

[This corrects the article DOI: 10.3389/fcvm.2022.892861.].

Accelerated Cardiac Aging in Patients With Congenital Heart Disease.

An increasing number of patients with congenital heart disease (CHD) survive into adulthood but develop long-term complications including heart failure (HF). Cellular senescence, classically defined as stable cell cycle arrest, is implicated in biological processes such as embryogenesis, wound healing, and aging. Senescent cells have a complex senescence-associated secretory phenotype (SASP), involving a range of pro-inflammatory factors with important paracrine and autocrine effects on cell and tissue biology. While senescence has been mainly considered as a cause of diseases in the adulthood, it may be also implicated in some of the poor outcomes seen in patients with complex CHD. We propose that patients with CHD suffer from multiple repeated stress from an early stage of the life, which wear out homeostatic mechanisms and cause premature cardiac aging, with this term referring to the time-related irreversible deterioration of the organ physiological functions and integrity. In this review article, we gathered evidence from the literature indicating that growing up with CHD leads to abnormal inflammatory response, loss of proteostasis, and precocious age in cardiac cells. Novel research on this topic may inspire new therapies preventing HF in adult CHD patients.

Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pulmonary Artery Reconstruction in Growing Piglets.

Surgical treatment of congenital heart defects affecting the right ventricular outflow tract often requires complex reconstruction and multiple reoperations. With a randomized controlled trial, we compared a novel tissue-engineered small intestine submucosa-based graft for pulmonary artery reconstruction (seeded with mesenchymal stem cells derived from Wharton's Jelly) with conventional small intestine submucosa in growing piglets. Six months after implantation, seeded grafts showed integration with host tissues at cellular level and exhibited growth potential on transthoracic echocardiography and cardiovascular magnetic resonance. Our seeded graft is a promising biomaterial for pulmonary artery reconstruction in pediatric patients with right ventricular outflow tract abnormalities.

Allogeneic Mesenchymal Stromal Cell Injection to Alleviate Ischemic Heart Failure Following Arterial Switch Operation.

Cell therapy is a promising tool to prevent and treat heart failure in congenital heart disease. We report the first case of intramyocardial injection of allogeneic mesenchymal stromal cells as rescue therapy in a neonate with ischemic heart failure following arterial switch procedure for isolated transposition of the great arteries. (Level of Difficulty: Advanced.).

ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation.

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell's function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol-nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including SLC25A1-which encodes the mitochondrial citrate carrier-and ACLY, thus promoting the existence of a proinflammatory loop involving SLC25A1 and ACLY genes.

Phenolic Compounds of Red Wine Aglianico del Vulture Modulate the Functional Activity of Macrophages via Inhibition of NF-κB and the Citrate Pathway.

Phenolic compounds of red wine powder (RWP) extracted from the Italian red wine Aglianico del Vulture have been investigated for the potential immunomodulatory and anti-inflammatory capacity on human macrophages. These compounds reduce the secretion of IL-1ß, IL-6, and TNF-α proinflammatory cytokines and increase the release of IL-10 anti-inflammatory cytokine induced by lipopolysaccharide (LPS). In addition, RWP restores Annexin A1 levels, thus involving activation of proresolutive pathways. Noteworthy, RWP lowers NF-κB protein levels, promoter activity, and nuclear translocation. As a consequence of NF-κB inhibition, reduced promoter activities of SLC25A1-encoding the mitochondrial citrate carrier (CIC)-and ATP citrate lyase (ACLY) metabolic genes have been observed. CIC, ACLY, and citrate are components of the citrate pathway: in LPS-activated macrophages, the mitochondrial citrate is exported by CIC into the cytosol where it is cleaved by ACLY in oxaloacetate and acetyl-CoA, precursors for ROS, NO·, and PGE2 inflammatory mediators. We identify the citrate pathway as a RWP target in carrying out its anti-inflammatory activity since RWP reduces CIC and ACLY protein levels, ACLY enzymatic activity, the cytosolic citrate concentration, and in turn ROS, NO·, PGE2, and histone acetylation levels. Overall findings suggest that RWP potentially restores macrophage homeostasis by suppressing inflammatory pathways and activating proresolutive processes.

Reconstruction of the Swine Pulmonary Artery Using a Graft Engineered With Syngeneic Cardiac Pericytes.

The neonatal heart represents an attractive source of regenerative cells. Here, we report the results of a randomized, controlled, investigator-blinded preclinical study, which assessed the safety and effectiveness of a matrix graft cellularized with cardiac pericytes (CPs) in a piglet model of pulmonary artery (PA) reconstruction. Within each of five trios formed by 4-week-old female littermate piglets, one element (the donor) was sacrificed to provide a source of CPs, while the other two elements (the graft recipients) were allowed to reach the age of 10 weeks. During this time interval, culture-expanded donor CPs were seeded onto swine small intestinal submucosa (SIS) grafts, which were then shaped into conduits and conditioned in a flow bioreactor. Control unseeded SIS conduits were subjected to the same procedure. Then, recipient piglets were randomized to surgical reconstruction of the left PA (LPA) with unseeded or CP-seeded SIS conduits. Doppler echocardiography and cardiac magnetic resonance imaging (CMRI) were performed at baseline and 4-months post-implantation. Vascular explants were examined using histology and immunohistochemistry. All animals completed the scheduled follow-up. No group difference was observed in baseline imaging data. The final Doppler assessment showed that the LPA's blood flow velocity was similar in the treatment groups. CMRI revealed a mismatch in the average growth of the grafted LPA and contralateral branch in both treatment groups. Histology of explanted arteries demonstrated that the CP-seeded grafts had a thicker luminal cell layer, more intraparietal arterioles, and a higher expression of endothelial nitric oxide synthase (eNOS) compared with unseeded grafts. Moreover, the LPA stump adjacent to the seeded graft contained more elastin and less collagen than the unseeded control. Syngeneic CP engineering did not accomplish the primary goal of supporting the graft's growth but was able to improve secondary outcomes, such as the luminal cellularization and intraparietal vascularization of the graft, and elastic remodeling of the recipient artery. The beneficial properties of neonatal CPs may be considered in future bioengineering applications aiming to reproduce the cellular composition of native arteries.

Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pediatric Translation in Heart Defect.

The materials available for the right ventricular outflow tract (RVOT) reconstruction in patients with tetralogy of fallot (TOF)/pulmonary atresia come with the severe limitation of long-term degeneration and lack of growth potential, causing right ventricular dysfunction, aneurysm formation, and arrhythmias, thus necessitating several high-risk reoperations throughout patients' lives. In this study, we evaluated the capacity of mesenchymal stem cells (MSCs) derived from the Wharton's Jelly (WJ-MSCs), the gelatinous inner portion of the umbilical cord, to grow and recellularize an extracellular matrix (ECM) graft in our optimized xeno-free, good manufacturing practice-compliant culture system. WJ-MSCs were phenotypically and functionally characterized by flow cytometry and multilineage differentiation capacity, respectively. The typical MSC immunophenotype and functional characteristics were retained in our xeno-free culture system, as well as the capacity to grow and engraft onto a naturally occurring scaffold. WJ-MSCs, from both human and swine source, showed excellent capacity to recellularize ECM graft producing a living cell-seeded construct. In addition, we have provided an in vivo proof of concept of feasibility of the cellularized conduit, engineered with swine WJ-MSCs, to be used in a novel porcine model of main pulmonary artery reconstruction, where it showed good integration within the host tissue. Our study indicates that the addition of WJ-MSCs to the ECM scaffold can upgrade the material, converting it into a living tissue, with the potential to grow, repair, and remodel the RVOT. These results could potentially represent a paradigm shift in pediatric cardiac intervention toward new modalities for effective and personalized surgical restoration of pulmonary artery and RVOT function in TOF/pulmonary atresia patients. Impact Statement The materials available for pulmonary artery reconstruction in pediatric patients with Congenital Heart Defect come with the limitation of long-term degeneration and lack of growth, thus necessitating several reoperations. Here, we describe a novel approach combining perinatal stem cells and naturally occurring scaffold to create a living tissue engineered conduit that showed good growth potential in a pulmonary artery reconstruction porcine model. We envision this approach is of great interest and relevance in tissue engineering field applied to cardiovascular regenerative medicine, as it may open up new avenues for correction of congenital cardiac defects, with remarkable medical and social benefits.

COVID-19, State of the Adult and Pediatric Heart: From Myocardial Injury to Cardiac Effect of Potential Therapeutic Intervention.

While the COVID-19 pandemic continues to spread rapidly, resulting in considerable morbidity and mortality worldwide, multiple efforts are being made by the international scientific community to understand the pathogenesis of the viral infection and its clinical outcome. Older age and comorbidities have consistently been reported as risk factors for unfavorable prognosis, with cardiovascular disease accounting for up to 10 % of comorbid conditions among the infected patients. An understanding of the mechanism underlying the effect of this infection on patients with cardiovascular disease is essential to manage and improve clinical strategies against the disease in that population. In this review, we summarize the impact of COVID-19 on patients with underlying cardiovascular conditions and the cardiac implications of known and emerging therapeutic strategies. Our future effort will aim to further elucidate how the type and severity of the cardiac disease, with particular regard to Congenital Heart Disease, influences the prognosis and the outcome of the viral infection.

In Vitro and In Vivo Preclinical Testing of Pericyte-Engineered Grafts for the Correction of Congenital Heart Defects.

Background We have previously reported the possibility of using pericytes from leftovers of palliative surgery of congenital heart disease to engineer clinically certified prosthetic grafts. Methods and Results Here, we assessed the feasibility of using prosthetic conduits engineered with neonatal swine pericytes to reconstruct the pulmonary artery of 9-week-old piglets. Human and swine cardiac pericytes were similar regarding anatomical localization in the heart and antigenic profile following isolation and culture expansion. Like human pericytes, the swine surrogates form clones after single-cell sorting, secrete angiogenic factors, and extracellular matrix proteins and support endothelial cell migration and network formation in vitro. Swine pericytes seeded or unseeded (control) CorMatrix conduits were cultured under static conditions for 5 days, then they were shaped into conduits and incubated in a flow bioreactor for 1 or 2 weeks. Immunohistological studies showed the viability and integration of pericytes in the outer layer of the conduit. Mechanical tests documented a reduction in stiffness and an increase in strain at maximum load in seeded conduits in comparison with unseeded conduits. Control and pericyte-engineered conduits were then used to replace the left pulmonary artery of piglets. After 4 months, anatomical and functional integration of the grafts was confirmed using Doppler echography, cardiac magnetic resonance imaging, and histology. Conclusions These findings demonstrate the feasibility of using neonatal cardiac pericytes for reconstruction of small-size branch pulmonary arteries in a large animal model.

Changes in inflammation and oxidative stress signalling pathways in coarcted aorta triggered by bicuspid aortic valve and growth in young children.

Neonates with coarctation of the aorta (CoA) combined with a bicuspid aortic valve (BAV) show significant structural differences compared to neonatal CoA patients with a normal tricuspid aortic valve (TAV). These effects are likely to change over time in response to growth. This study investigated proteomic differences between coarcted aortic tissue of BAV and TAV patients in children older than one month. Aortic tissue just proximal to the coarctation site was collected from 10 children (BAV; n=6, 1.9±1.7 years, TAV; n=4, 1.7±1.5 years, (mean ± SEM, P=0.92.) Tissue were snap frozen, proteins extracted, and the extracts used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. A total of 1811 protein and 76 phosphoprotein accession numbers were detected, of which 40 proteins and 6 phosphoproteins were significantly differentially expressed between BAV and TAV patients. Several canonical pathways involved in inflammation demonstrated enriched protein expression, including acute phase response signalling, EIF2 signalling and macrophage production of IL12 and reactive oxygen species. Acute phase response signalling also demonstrated enriched phosphoprotein expression, as did Th17 activation. Other pathways with significantly enriched protein expression include degradation of superoxide radicals and several pathways involved in apoptosis. This work suggests that BAV CoA patients older than one month have an altered proteome consistent with changes in inflammation, apoptosis and oxidative stress compared to TAV CoA patients of the same age. There is no evidence of structural differences, suggesting the pathology associated with BAV evolves with age in paediatric CoA patients.

miR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183-KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183-KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.