Lymphatic endothelial transcription factor Tbx1 promotes an immunosuppressive microenvironment to facilitate post-myocardial infarction repair.

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge syndrome disease gene that encodes a T-box transcription factor (TF). We found induced profound lymphangiogenic and immunomodulatory gene expression changes in lymphatic endothelial cells (LECs) after myocardial infarction (MI). The activated LECs penetrated the infarcted area and functioned as intramyocardial immune hubs to increase the numbers of tolerogenic dendritic cells (tDCs) and regulatory T (Treg) cells through the chemokine Ccl21 and integrin Icam1, thereby inhibiting the expansion of autoreactive CD8+ T cells and promoting reparative macrophage expansion to facilitate post-MI repair. Mimicking its timing and implementation may be an additional approach to treating autoimmunity-mediated cardiac diseases.

Cellular nucleic acid binding protein facilitates cardiac repair after myocardial infarction by activating ß-catenin signaling.

The regenerative capacity of the adult mammalian heart is limited, while the neonatal heart is an organ with regenerative and proliferative ability. Activating adult cardiomyocytes (CMs) to re-enter the cell cycle is an effective therapeutic method for ischemic heart disease such as myocardial infarction (MI) and heart failure. Here, we aimed to reveal the role and potential mechanisms of cellular nucleic acid binding protein (CNBP) in cardiac regeneration and repair after heart injury. CNBP is highly expressed within 7 days post-birth while decreases significantly with the loss of regenerative ability. In vitro, overexpression of CNBP promoted CM proliferation and survival, whereas knockdown of CNBP inhibited these processes. In vivo, knockdown of CNBP in CMs robustly hindered myocardial regeneration after apical resection in neonatal mice. In adult MI mice, CM-specific CNBP overexpression in the infarct border zone ameliorated myocardial injury in acute stage and facilitated CM proliferation and functional recovery in the long term. Quantitative proteomic analysis with TMT labeling showed that CNBP overexpression promoted the DNA replication, cell cycle progression, and cell division. Mechanically, CNBP overexpression increased the expression of ß-catenin and its downstream target genes CCND1 and c-myc; Furthermore, Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays showed that CNBP could directly bind to the ß-catenin promoter and promote its transcription. CNBP also upregulated the expression of G1/S-related cell cycle genes CCNE1, CDK2, and CDK4. Collectively, our study reveals the positive role of CNBP in promoting cardiac repair after injury, providing a new therapeutic option for the treatment of MI.

At the heart of inflammation: Unravelling cardiac resident macrophage biology.

Cardiovascular disease remains one of the leading causes of death globally. Recent advancements in sequencing technologies have led to the identification of a unique population of macrophages within the heart, termed cardiac resident macrophages (CRMs), which exhibit self-renewal capabilities and play crucial roles in regulating cardiac homeostasis, inflammation, as well as injury and repair processes. This literature review aims to elucidate the origin and phenotypic characteristics of CRMs, comprehensively outline their contributions to cardiac homeostasis and further summarize their functional roles and molecular mechanisms implicated in the onset and progression of cardiovascular diseases. These insights are poised to pave the way for novel therapeutic strategies centred on targeted interventions based on the distinctive properties of resident macrophages.

Current optimized strategies for stem cell-derived extracellular vesicle/exosomes in cardiac repair.

Ischemic heart diseases remain the leading cause of death globally, and stem cell-based therapy has been investigated as a potential approach for cardiac repair. Due to poor survival and engraftment in the cardiac ischemic milieu post transplantation, the predominant therapeutic effects of stem cells act via paracrine actions, by secreting extracellular vesicles (EVs) and/or other factors. Exosomes are nano-sized EVs of endosomal origin, and now viewed as a major contributor in facilitating myocardial repair and regeneration. However, EV/exosome therapy has major obstacles before entering clinical settings, such as limited production yield, unstable biological activity, poor homing efficiency, and low tissue retention. This review aims to provide an overview of the biogenesis and mechanisms of stem cell-derived EV/exosomes in the process of cardiac repair and discuss the current advancements in different optimized strategies to produce high-yield EV/exosomes with higher bioactivity, or engineer them with improved homing efficiency and therapeutic potency. In particular, we outline recent findings toward preclinical and clinical translation of EV/exosome therapy in ischemic heart diseases, and discuss the potential barriers in regard to clinical translation of EV/exosome therapy.

Adenosine kinase promotes post-infarction cardiac repair by epigenetically maintaining reparative macrophage phenotype.

Pro-inflammatory and reparative macrophages are crucial in clearing necrotic myocardium and promoting cardiac repair after myocardial infarction (MI), respectively. Extracellular adenosine has been demonstrated to modulate macrophage polarization through adenosine receptors. However, the role of intracellular adenosine in macrophage polarization has not been explored and adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels. Here, we aimed to elucidate the role of ADK in macrophage polarization and its subsequent impact on MI. We demonstrated that ADK was upregulated in bone marrow-derived macrophages (BMDMs) after IL-4 treatment and was highly expressed in the infarct area at day 7 post-MI, especially in macrophages. Compared with wild-type mice, myeloid-specific Adk knockout mice showed increased infarct size, limited myofibroblast differentiation, reduced collagen deposition and more severe cardiac dysfunction after MI, which was related to impaired reparative macrophage phenotype in MI tissue. We found that ADK deletion or inhibition significantly decreased the expression of reparative genes, such as Arg1, Ym1, Fizz1, and Cd206 in BMDMs after IL-4 treatment. The increased intracellular adenosine due to Adk deletion inhibited transmethylation reactions and decreased the trimethylation of H3K4 in BMDMs after IL-4 treatment. Mechanistically, we demonstrated that Adk deletion suppressed reparative macrophage phenotype through decreased IRF4 expression, which resulted from reduced levels of H3K4me3 on the Irf4 promotor. Together, our study reveals that ADK exerts a protective effect against MI by promoting reparative macrophage polarization through epigenetic mechanisms.

Application of Nanomaterials in Stem Cell-Based Therapeutics for Cardiac Repair and Regeneration.

Cardiovascular disease is a leading cause of disability and death worldwide. Although the survival rate of patients with heart diseases can be improved with contemporary pharmacological treatments and surgical procedures, none of these therapies provide a significant improvement in cardiac repair and regeneration. Stem cell-based therapies are a promising approach for functional recovery of damaged myocardium. However, the available stem cells are difficult to differentiate into cardiomyocytes, which result in the extremely low transplantation efficiency. Nanomaterials are widely used to regulate the myocardial differentiation of stem cells, and play a very important role in cardiac tissue engineering. This study discusses the current status and limitations of stem cells and cell-derived exosomes/micro RNAs based cardiac therapy, describes the cardiac repair mechanism of nanomaterials, summarizes the recent advances in nanomaterials used in cardiac repair and regeneration, and evaluates the advantages and disadvantages of the relevant nanomaterials. Besides discussing the potential clinical applications of nanomaterials in cardiac therapy, the perspectives and challenges of nanomaterials used in stem cell-based cardiac repair and regeneration are also considered. Finally, new research directions in this field are proposed, and future research trends are highlighted.

Insight into Heart-Tailored Architectures of Hydrogel to Restore Cardiac Functions after Myocardial Infarction.

With permanent heart muscle injury or death, myocardial infarction (MI) is complicated by inflammatory, proliferation and remodeling phases from both the early ischemic period and subsequent infarct expansion. Though in situ re-establishment of blood flow to the infarct zone and delays of the ventricular remodeling process are current treatment options of MI, they fail to address massive loss of viable cardiomyocytes while transplanting stem cells to regenerate heart is hindered by their poor retention in the infarct bed. Equipped with heart-specific mimicry and extracellular matrix (ECM)-like functionality on the network structure, hydrogels leveraging tissue-matching biomechanics and biocompatibility can mechanically constrain the infarct and act as localized transport of bioactive ingredients to refresh the dysfunctional heart under the constant cyclic stress. Given diverse characteristics of hydrogel including conductivity, anisotropy, adhesiveness, biodegradability, self-healing and mechanical properties driving local cardiac repair, we aim to investigate and conclude the dynamic balance between ordered architectures of hydrogels and the post-MI pathological milieu. Additionally, our review summarizes advantages of heart-tailored architectures of hydrogels in cardiac repair following MI. Finally, we propose challenges and prospects in clinical translation of hydrogels to draw theoretical guidance on cardiac repair and regeneration after MI.

Fabrication of blended nanofibrous cardiac patch transplanted with TGF-ß3 and human umbilical cord MSCs-derived exosomes for potential cardiac regeneration after acute myocardial infarction.

Acute myocardial infarction (AMI) is a common cardiovascular condition that progressively results in heart failure. In the present study, we have designed to load transforming growth factor beta 3 (TGF-ß3) and cardio potential exosomes into the blended polycaprolactone/type I collagen (PCL/COL-1) nanofibrous patch (Exo@TGF-ß3@NFs) and examined its feasibility for cardiac repair. The bioactivity of the developed NFs towards the migration and proliferation of human umbilical vein endothelial cells was determined using in vitro cell compatibility assays. Additionally, Exo@TGF-ß3/NFs showed up-regulation of genes involved in angiogenesis and mesenchymal differentiations in vitro. The in vivo experiments performed 4 weeks after transplantation showed that the Exo@TGF-ß3@NFs had a higher LV ejection fraction and fraction shortening functions. Subsequently, it has been determined that Exo@TGF-ß3@NFs significantly reduced AMI size and fibrosis and increased scar thickness. The developed NFs approach will become a useful therapeutic approach for the treatment of AMI.

Regulation of Cardiomyocyte Division During Cardiac Regeneration.

PURPOSE OF REVIEW: This review explores efforts made over the previous three decades to determine mechanisms of cardiomyocyte cell division. Many investigators have explored cell therapy strategies in animal models and clinical trials over the past 2 decades with marginal results thus far in clinical testing. Hence, there is a greater focus now on strategies to induce cardiomyocyte proliferation. RECENT FINDINGS: Reports to induce reactivation of the cardiomyocyte cell cycle predated the focus on cell therapy, and we summarize the literature on this topic, which began with the very first transgenic mouse studies in cardiovascular science. These earlier studies form the foundation for the use of cell cycle manipulation in cardiac repair and should inform current and future investigations with respect to rigor of assessment in the degree of cardiomyocyte cell division and gold standard measures of cardiac functional improvement.

Immune Cells in Cardiac Injury Repair and Remodeling.

PURPOSE OF REVIEW: Immune cells are emerging as central cellular components of the heart which communicate with cardiac resident cells during homeostasis, cardiac injury, and remodeling. These findings are contributing to the development and continuous expansion of the new field of cardio-immunology. We review the most recent literature on this topic and discuss ongoing and future efforts to advance this field forward. RECENT FINDINGS: Cell-fate mapping, strategy depleting, and reconstituting immune cells in pre-clinical models of cardiac disease, combined with the investigation of the human heart at the single cell level, are contributing immensely to our understanding of the complex intercellular communication between immune and non-immune cells in the heart. While the acute immune response is necessary to initiate inflammation and tissue repair post injury, it becomes detrimental when sustained over time and contributes to adverse cardiac remodeling and pathology. Understanding the specific functions of immune cells in the context of the cardiac environment will provide new opportunities for immunomodulation to induce or tune down inflammation as needed in heart disease.

Hydrogels for Cardiac Restorative Support: Relevance of Gelation Mechanisms for Prospective Clinical Use.

PURPOSE OF REVIEW: Cardiac tissue regenerative strategies have gained much traction over the years, in particular those utilizing hydrogels. With our review, and with special focus on supporting post-myocardial infarcted tissue, we aim to provide insights in determining crucial design considerations of a hydrogel and the implications these could have for future clinical use. RECENT FINDINGS: To date, two hydrogel delivery strategies are being explored, cardiac injection or patch, to treat myocardial infarction. Recent advances have demonstrated that the mechanism by which a hydrogel is gelated (i.e., physically or chemically cross-linked) not only impacts the biocompatibility, mechanical properties, and chemical structure, but also the route of delivery of the hydrogel and thus its effect on cardiac repair. With regard to cardiac regeneration, various hydrogels have been developed with the ability to function as a delivery system for therapeutic strategies (e.g., drug and stem cells treatments), as well as a scaffold to guide cardiac tissue regeneration following myocardial infarction. However, these developments remain within the experimental and pre-clinical realm and have yet to transition towards the clinical setting.

New Insights into the Reparative Angiogenesis after Myocardial Infarction.

Myocardial infarction (MI) causes massive loss of cardiac myocytes and injury to the coronary microcirculation, overwhelming the limited capacity of cardiac regeneration. Cardiac repair after MI is finely organized by complex series of procedures involving a robust angiogenic response that begins in the peri-infarcted border area of the infarcted heart, concluding with fibroblast proliferation and scar formation. Efficient neovascularization after MI limits hypertrophied myocytes and scar extent by the reduction in collagen deposition and sustains the improvement in cardiac function. Compelling evidence from animal models and classical in vitro angiogenic approaches demonstrate that a plethora of well-orchestrated signaling pathways involving Notch, Wnt, PI3K, and the modulation of intracellular Ca2+ concentration through ion channels, regulate angiogenesis from existing endothelial cells (ECs) and endothelial progenitor cells (EPCs) in the infarcted heart. Moreover, cardiac repair after MI involves cell-to-cell communication by paracrine/autocrine signals, mainly through the delivery of extracellular vesicles hosting pro-angiogenic proteins and non-coding RNAs, as microRNAs (miRNAs). This review highlights some general insights into signaling pathways activated under MI, focusing on the role of Ca2+ influx, Notch activated pathway, and miRNAs in EC activation and angiogenesis after MI.

Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

Neonatal Cardiovascular-Progenitor-Cell-Derived Extracellular Vesicles Activate YAP1 in Adult Cardiac Progenitor Cells.

New stem cell and extracellular-vesicle-based therapies have the potential to improve outcomes for the increasing number of patients with heart failure. Since neonates have a significantly enhanced regenerative ability, we hypothesized that extracellular vesicles isolated from Islet-1+ expressing neonatal human cardiovascular progenitors (CPCs) will induce transcriptomic changes associated with improved regenerative capability when co-cultured with CPCs derived from adult humans. In order to test this hypothesis, we isolated extracellular vesicles from human neonatal Islet-1+ CPCs, analyzed the extracellular vesicle content using RNAseq, and treated adult CPCs with extracellular vesicles derived from neonatal CPCs to assess their functional effect. AKT, ERBB, and YAP1 transcripts were elevated in adult CPCs treated with neonatal CPC-derived extracellular vesicles. YAP1 is lost after the neonatal period but can stimulate cardiac regeneration. Our results demonstrate that YAP1 and additional transcripts associated with improved cardiovascular regeneration, as well as the activation of the cell cycle, can be achieved by the treatment of adult CPCs with neonatal CPC-derived extracellular vesicles. Progenitor cells derived from neonates secrete extracellular vesicles with the potential to stimulate and potentially improve functional effects in adult CPCs used for cardiovascular repair.

Human engineered heart tissue transplantation in a guinea pig chronic injury model.

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 106 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach.

Characterizing the transition from immune response to tissue repair after myocardial infarction by multiparametric imaging.

Persistent inflammation following myocardial infarction (MI) precipitates adverse outcome including acute ventricular rupture and chronic heart failure. Molecular imaging allows longitudinal assessment of immune cell activity in the infarct territory and predicts severity of remodeling. We utilized a multiparametric imaging platform to assess the immune response and cardiac healing following MI in mice. Suppression of circulating macrophages prior to MI paradoxically resulted in higher total leukocyte content in the heart, demonstrated by increased CXC motif chemokine receptor 4 (CXCR4) positron emission tomography imaging. This supported the formation of a thrombus overlying the injured region, as identified by magnetic resonance imaging. The injured and thrombotic region in macrophage depeleted mice subsequently showed active calcification, as evidenced by accumulation of 18F-fluoride and by cardiac computed tomography. Importantly, macrophage suppression triggered a prolonged inflammatory response confirmed by post-mortem tissue analysis that was associated with higher mortality from ventricular rupture early after occlusion and with increased infarct size and worse chronic contractile function at 6 weeks after reperfusion. These findings establish a molecular imaging toolbox for monitoring the interplay between adverse immune response and tissue repair after MI. This may serve as a foundation for development and monitoring of novel targeted therapies that may include immune modulation and endogenous healing support.

A deep dive into the darning effects of biomaterials in infarct myocardium: current advances and future perspectives.

Myocardial infarction (MI) occurs due to the obstruction of coronary arteries, a major crux that restricts blood flow and thereby oxygen to the distal part of the myocardium, leading to loss of cardiomyocytes and eventually, if left untreated, leads to heart failure. MI, a potent cardiovascular disorder, requires intense therapeutic interventions and thereby presents towering challenges. Despite the concerted efforts, the treatment strategies for MI are still demanding, which has paved the way for the genesis of biomaterial applications. Biomaterials exhibit immense potentials for cardiac repair and regeneration, wherein they act as extracellular matrix replacing scaffolds or as delivery vehicles for stem cells, protein, plasmids, etc. This review concentrates on natural, synthetic, and hybrid biomaterials; their function; and interaction with the body, mechanisms of repair by which they are able to improve cardiac function in a MI milieu. We also provide focus on future perspectives that need attention. The cognizance provided by the research results certainly indicates that biomaterials could revolutionize the treatment paradigms for MI with a positive impact on clinical translation.

Multicellular regulation of miR-196a-5p and miR-425-5 from adipose stem cell-derived exosomes and cardiac repair.

Cardiac transplantation of adipose-derived stem cells (ASC) modulates the post-myocardial infarction (post-MI) repair response. Biomolecules secreted or shuttled within extracellular vesicles, such as exosomes, may participate in the concerted response. We investigated the exosome's microRNAs due to their capacity to fine-tune gene expression, potentially affecting the multicellular repair response. We profiled and quantified rat ASC-exosome miRNAs and used bioinformatics to select uncharacterized miRNAs down-regulated in post-MI related to cardiac repair. We selected and validated miR-196a-5p and miR-425-5p as candidates for the concerted response in neonatal cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages using a high-content screening platform. Both miRNAs prevented cardiomyocyte ischemia-induced mitochondrial dysfunction and reactive oxygen species production, increased angiogenesis, and polarized macrophages toward the anti-inflammatory M2 immunophenotype. Moreover, miR-196a-5p reduced and reversed myofibroblast activation and decreased collagen expression. Our data provide evidence that the exosome-derived miR-196a-5p and miR-425-5p influence biological processes critical to the concerted multicellular repair response post-MI.

Oxidant stress-sensitive circRNA Mdc1 controls cardiomyocyte chromosome stability and cell cycle re-entry during heart regeneration.

Targeting cardiomyocyte plasticity has emerged as a new strategy for promoting heart repair after myocardial infarction. However, the precise mechanistic network underlying heart regeneration is not completely understood. As noncoding RNAs, circular RNAs (circRNAs) play essential roles in regulating cardiac physiology and pathology. The present study aimed to investigate the potential roles of circMdc1 in cardiac repair after injury and elucidate its underlying mechanisms. Here, we identified that circMdc1 levels were upregulated in postnatal mouse hearts but downregulated in the regenerative myocardium. The expression of circMdc1 in cardiomyocytes is sensitive to oxidative stress, which was attenuated by N-acetyl-cysteine. Enforced circMdc1 expression inhibited cardiomyocyte proliferation, while circMdc1 silencing led to cardiomyocyte cell cycle re-entry. In vivo, the cardiac-specific adeno-associated virus-mediated knockdown of circMdc1 promoted cardiac regeneration and heart repair accompanied by improved heart function. Conversely, circMdc1 overexpression blunted the regenerative capacity of neonatal hearts after apex resection. Moreover, circMdc1 was able to block the translation of its host gene Mdc1 specifically by binding to PABP, affecting DNA damage and the chromosome stability of cardiomyocytes. Furthermore, overexpression of Mdc1 caused damaged mouse hearts to regenerate and repair after myocardial infarction in vivo. Oxidative stress-sensitive circMdc1 plays an important role in cardiac regeneration and heart repair after injury by regulating DNA damage and chromosome stability in cardiomyocytes by blocking the translation of the host gene Mdc1.

Direct reprogramming induces vascular regeneration post muscle ischemic injury.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFß, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.