Direct conversion of cardiac fibroblasts into endothelial-like cells using Sox17 and Erg.

Endothelial cells are a heterogeneous population with various organ-specific and conserved functions that are critical to organ development, function, and regeneration. Here we report a Sox17-Erg direct reprogramming approach that uses cardiac fibroblasts to create differentiated endothelial cells that demonstrate endothelial-like molecular and physiological functions in vitro and in vivo. Injection of these induced endothelial cells into myocardial infarct sites after injury results in improved vascular perfusion of the scar region. Furthermore, we use genomic analyses to illustrate that Sox17-Erg reprogramming instructs cardiac fibroblasts toward an arterial-like identity. This results in a more efficient direct conversion of fibroblasts into endothelial-like cells when compared to traditional Etv2-based reprogramming. Overall, this Sox17-Erg direct reprogramming strategy offers a robust tool to generate endothelial cells both in vitro and in vivo, and has the potential to be used in repairing injured tissue.

Biomimetic Nano-Degrader Based CD47-SIRPα Immune Checkpoint Inhibition Promotes Macrophage Efferocytosis for Cardiac Repair.

CD47-SIRPα axis is an immunotherapeutic target in tumor therapy. However, current monoclonal antibody targeting CD47-SIRPα axis is associated with on-target off-tumor and antigen sink effects, which significantly limit its potential clinical application. Herein, a biomimetic nano-degrader is developed to inhibit CD47-SIRPα axis in a site-specific manner through SIRPα degradation, and its efficacy in acute myocardial infarction (AMI) is evaluated. The nano-degrader is constructed by hybridizing liposome with red blood cell (RBC) membrane (RLP), which mimics the CD47 density of senescent RBCs and possesses a natural high-affinity binding capability to SIRPα on macrophages without signaling capacity. RLP would bind with SIRPα and induce its lysosomal degradation through receptor-mediated endocytosis. To enhance its tissue specificity, Ly6G antibody conjugation (aRLP) is applied, enabling its attachment to neutrophils and accumulation within inflammatory sites. In the myocardial infarction model, aRLP accumulated in the infarcted myocardium blocks CD47-SIRPα axis and subsequently promoted the efferocytosis of apoptotic cardiomyocytes by macrophage, improved heart repair. This nano-degrader efficiently degraded SIRPα in lysosomes, providing a new strategy for immunotherapy with great clinical transformation potential.

Fibroblast Reprogramming in Cardiac Repair.

Cardiovascular disease is one of the major causes of death worldwide. Limited proliferative capacity of adult mammalian cardiomyocytes has prompted researchers to exploit regenerative therapy after myocardial injury, such as myocardial infarction, to attenuate heart dysfunction caused by such injury. Direct cardiac reprogramming is a recently emerged promising approach to repair damaged myocardium by directly converting resident cardiac fibroblasts into cardiomyocyte-like cells. The achievement of in vivo direct reprogramming of fibroblasts has been shown, by multiple laboratories independently, to improve cardiac function and mitigate fibrosis post-myocardial infarction, which holds great potential for clinical application. There have been numerous pieces of valuable work in both basic and translational research to enhance our understanding and continued refinement of direct cardiac reprogramming in recent years. However, there remain many challenges to overcome before we can truly take advantage of this technique to treat patients with ischemic cardiac diseases. Here, we review recent progress of fibroblast reprogramming in cardiac repair, including the optimization of several reprogramming strategies, mechanistic exploration, and translational efforts, and we make recommendations for future research to further understand and translate direct cardiac reprogramming from bench to bedside. Challenges relating to these efforts will also be discussed.

Genetically Engineered Macrophages Co-Loaded with CD47 Inhibitors Synergistically Reconstruct Efferocytosis and Improve Cardiac Remodeling Post Myocardial Ischemia Reperfusion Injury.

Efferocytosis, mediated by the macrophage receptor MerTK (myeloid-epithelial-reproductive tyrosine kinase), is a significant contributor to cardiac repair after myocardial ischemia-reperfusion (MI/R) injury. However, the death of resident cardiac macrophages (main effector cells), inactivation of MerTK （main effector receptor), and overexpression of "do not eat me" signals (brake signals, such as CD47), collectively lead to the impediment of efferocytosis in the post-MI/R heart. To date, therapeutic strategies targeting individual above obstacles are relatively lacking, let alone their effectiveness being limited due to constraints from the other concurrent two. Herein, inspired by the application research of chimeric antigen receptor macrophages (CAR-Ms) in solid tumors, a genetically modified macrophage-based synergistic drug delivery strategy that effectively challenging the three major barriers in an integrated manner is developed. This strategy involves the overexpression of exogenous macrophages with CCR2 (C-C chemokine receptor type 2) and cleavage-resistant MerTK, as well as surface clicking with liposomal PEP-20 (a CD47 antagonist). In MI/R mice model, this synergistic strategy can effectively restore cardiac efferocytosis after intravenous injection, thereby alleviating the inflammatory response, ultimately preserving cardiac function. This therapy focuses on inhibiting the initiation and promoting active resolution of inflammation, providing new insights for immune-regulatory therapy.

Hippo pathway-manipulating neutrophil-mimic hybrid nanoparticles for cardiac ischemic injury via modulation of local immunity and cardiac regeneration.

The promise of regeneration therapy for restoration of damaged myocardium after cardiac ischemic injury relies on targeted delivery of proliferative molecules into cardiomyocytes whose healing benefits are still limited owing to severe immune microenvironment due to local high concentration of proinflammatory cytokines. Optimal therapeutic strategies are therefore in urgent need to both modulate local immunity and deliver proliferative molecules. Here, we addressed this unmet need by developing neutrophil-mimic nanoparticles NM@miR, fabricated by coating hybrid neutrophil membranes with artificial lipids onto mesoporous silica nanoparticles (MSNs) loaded with microRNA-10b. The hybrid membrane could endow nanoparticles with strong capacity to migrate into inflammatory sites and neutralize proinflammatory cytokines and increase the delivery efficiency of microRNA-10b into adult mammalian cardiomyocytes (CMs) by fusing with cell membranes and leading to the release of MSNs-miR into cytosol. Upon NM@miR administration, this nanoparticle could home to the injured myocardium, restore the local immunity, and efficiently deliver microRNA-10b to cardiomyocytes, which could reduce the activation of Hippo-YAP pathway mediated by excessive cytokines and exert the best proliferative effect of miR-10b. This combination therapy could finally improve cardiac function and mitigate ventricular remodeling. Consequently, this work offers a combination strategy of immunity modulation and proliferative molecule delivery to boost cardiac regeneration after injury.

Translational landscape of direct cardiac reprogramming reveals a role of Ybx1 in repressing cardiac fate acquisition.

Direct reprogramming of fibroblasts into induced cardiomyocytes holds great promise for heart regeneration. Although considerable progress has been made in understanding the transcriptional and epigenetic mechanisms of iCM reprogramming, its translational regulation remains largely unexplored. Here, we characterized the translational landscape of iCM reprogramming through integrative ribosome and transcriptomic profiling, and found extensive translatome repatterning during this process. Loss of function screening for translational regulators uncovered Ybx1 as a critical barrier to iCM induction. In a mouse model of myocardial infarction, removing Ybx1 enhanced in vivo reprogramming, resulting in improved heart function and reduced scar size. Mechanistically, Ybx1 depletion de-repressed the translation of its direct targets SRF and Baf60c, both of which mediated the effect of Ybx1 depletion on iCM generation. Furthermore, removal of Ybx1 allowed single factor Tbx5-mediated iCM conversion. In summary, this study revealed a new layer of regulatory mechanism that controls cardiac reprogramming at the translational level.

Temporal link prediction via adjusted sigmoid function and 2-simplex structure.

Temporal network link prediction is an important task in the field of network science, and has a wide range of applications in practical scenarios. Revealing the evolutionary mechanism of the network is essential for link prediction, and how to effectively utilize the historical information for temporal links and efficiently extract the high-order patterns of network structure remains a vital challenge. To address these issues, in this paper, we propose a novel temporal link prediction model with adjusted sigmoid function and 2-simplex structure (TLPSS). The adjusted sigmoid decay mode takes the active, decay and stable states of edges into account, which properly fits the life cycle of information. Moreover, the latent matrix sequence is introduced, which is composed of simplex high-order structure, to enhance the performance of link prediction method since it is highly feasible in sparse network. Combining the life cycle of information and simplex high-order structure, the overall performance of TLPSS is achieved by satisfying the consistency of temporal and structural information in dynamic networks. Experimental results on six real-world datasets demonstrate the effectiveness of TLPSS, and our proposed model improves the performance of link prediction by an average of 15% compared to other baseline methods.

Targeted delivery and ROS-responsive release of Resolvin D1 by platelet chimeric liposome ameliorates myocardial ischemia-reperfusion injury.

Resolvin D1 (RvD1) has been shown to provide effective protection against ischemia-reperfusion injury in multiple vital organs such as the heart, brain, kidney. However, the clinical translational potential of systemic administration of RvD1 in the treatment of ischemia-reperfusion injury is greatly limited due to biological instability and lack of targeting ability. Combining the natural inflammatory response and reactive oxygen species (ROS) overproduction after reperfusion injury, we developed a platelet-bionic, ROS-responsive RvD1 delivery platform. The resulting formulation enables targeted delivery of RvD1 to the injury site by hijacking circulating chemotactic monocytes, while achieving locally controlled release. In a mouse model of myocardial ischemia repefusuin (MI/R) injury, intravenous injection of our formula resulted in the enrichment of RvD1 in the injured area, which in turn promotes clearance of dead cells, production of specialized proresolving mediators (SPMs), and angiogenesis during injury repair, effectively improving cardiac function. This delivery system integrates drug bio-protection, targeted delivery and controlled release, which endow it with great clinical translational value.

Targeted neutrophil-mimetic liposomes promote cardiac repair by adsorbing proinflammatory cytokines and regulating the immune microenvironment.

Acute myocardial infarction (MI) induces a sterile inflammatory response that may result in poor cardiac remodeling and dysfunction. Despite the progress in anti-cytokine biologics, anti-inflammation therapy of MI remains unsatisfactory, due largely to the lack of targeting and the complexity of cytokine interactions. Based on the nature of inflammatory chemotaxis and the cytokine-binding properties of neutrophils, we fabricated biomimetic nanoparticles for targeted and broad-spectrum anti-inflammation therapy of MI. By fusing neutrophil membranes with conventional liposomes, we fabricated biomimetic liposomes (Neu-LPs) that inherited the surface antigens of the source cells, making them ideal decoys of neutrophil-targeted biological molecules. Based on their abundant chemokine and cytokine membrane receptors, Neu-LPs targeted infarcted hearts, neutralized proinflammatory cytokines, and thus suppressed intense inflammation and regulated the immune microenvironment. Consequently, Neu-LPs showed significant therapeutic efficacy by providing cardiac protection and promoting angiogenesis in a mouse model of myocardial ischemia-reperfusion. Therefore, Neu-LPs have high clinical translation potential and could be developed as an anti-inflammatory agent to remove broad-spectrum inflammatory cytokines during MI and other neutrophil-involved diseases.

Targeted immunomodulation therapy for cardiac repair by platelet membrane engineering extracellular vesicles via hitching peripheral monocytes.

Immune regulation therapies have been considered promising in the treatment of myocardial ischemia reperfusion (MI/R) injury. Mesenchymal stem cells derived extracellular vesicles (MSC-EVs) are of great potential for immune modulation by reprogramming macrophages but their therapeutic efficacy is hindered by insufficient targeting ability in vivo. Herein, we introduced the platelet membrane modified EVs (P-EVs) based on membrane fusion method to mimic the binding ability of platelets to monocytes. In the mouse model of MI/R injury, the intravenously injected P-EVs were mainly carried by circulating monocytes into the ischemic myocardium. In the inflammatory microenvironment, those monocytes subsequently differentiated into macrophages with enhanced phagocytosis, which probably promoted in-situ endocytosis of the superficial P-EVs by monocytes differentiated macrophages in large quantities. Then, the P-EVs successfully escaped from the macrophage lysosome and released the functional microRNAs (miRNAs) into the cytosol which facilitated the inflammatory macrophages (M1 phenotype) reprogramming to reparative macrophages (M2 phenotype). Finally, the immune microenvironment was regulated to realize cardiac repair. Thus, we supposed that the most likely delivery method was that monocytes mediated P-EVs migration into ischemic myocardium where P-EVs were mainly in-situ endocytosed by monocytes derived macrophages, which holds potential for immunoregulation on MI/R and other immune-related diseases in the future.

Direct in vivo reprogramming with non-viral sequential targeting nanoparticles promotes cardiac regeneration.

microRNA-mediated direct cardiac reprogramming, directly converts fibroblasts into induced cardiomyocyte-like cells (iCMs), which holds great promise in cardiac regeneration therapy. However, effective approaches to deliver therapeutic microRNA into cardiac fibroblasts (CFs) to induce in vivo cardiac reprogramming remain to be explored. Herein, a non-viral biomimetic system to directly reprogram CFs for cardiac regeneration after myocardial injury was developed by coating FH peptide-modified neutrophil-mimicking membranes on mesoporous silicon nanoparticles (MSNs) loaded with microRNA1, 133, 208, and 499 (miR Combo). Through utilizing the natural inflammation-homing ability of neutrophil membrane protein and FH peptide's high affinity to tenascin-C (TN-C) produced by CFs, this nanoparticle could realize sequential targeting to CFs in the injured heart and precise intracellular delivery of miRCombo, which induced reprogramming resident CFs into iCMs. In a mouse model of myocardial ischemia/reperfusion injury, intravenous injection of the nanoparticles successfully delivered miRCombo into fibroblasts and led to efficient reprogramming, resulting in improved cardiac function and attenuated fibrosis. This delivery system is minimally invasive and bio-safe, providing a proof-of-concept for biomimetic and sequential targeting nanomedicine delivery system for microRNA-mediated reprogramming therapy in multiple diseases.

Platelet-Like Fusogenic Liposome-Mediated Targeting Delivery of miR-21 Improves Myocardial Remodeling by Reprogramming Macrophages Post Myocardial Ischemia-Reperfusion Injury.

Inflammatory modulations focusing on macrophage phenotype are promising candidates to promote better cardiac healing post myocardial ischemia-reperfusion (MI/R) injury. However, the peak of monocyte/macrophage recruitment is later than the time when enhanced permeability and retention effect disappears, which greatly increases the difficulty of reprogramming macrophages through systemic administration. Meanwhile, the inability of nanomaterials to release their contents to specific intracellular locations through reasonable cellular internalization pathways is another obstacle to achieving macrophage reprogramming. Here, inspired by the increase in circulating platelet-monocyte aggregates in patients' post-MI/R and the high efficiency of fusogenic liposomes to deliver contents to the cytoplasm of target cells, a platelet-like fusogenic liposome (PLPs) is constructed. Under the coating of PLPs, mesoporous silica nanospheres with a payload of miR-21, an anti-inflammatory agent, can be specifically delivered to inflammatory monocytes in the blood circulation of MI/R induced mice. Then it directly enters the cytoplasm of monocytes through membrane fusion, thereby realizing the reparative reprogramming of the inflamed macrophages derived from it. In vivo administration of the resulting formula can effectively preserve the cardiac function of mice undergone MI/R. Minimal invasiveness and biological safety make this nano-platform a promising approach of immunotherapy.

Engineering extracellular vesicles with platelet membranes fusion enhanced targeted therapeutic angiogenesis in a mouse model of myocardial ischemia reperfusion.

Therapeutic angiogenesis is one promising strategy for the treatment of ischemic heart disease, which is the leading cause of death globally. In recent years, extracellular vesicles (EVs) have quickly gained much attention as a cell-free approach to stimulate angiogenesis. However, clinical applications of EVs are limited by their insufficient targeting capability. Herein, we introduce a method to enhance therapeutic angiogenesis based on platelet membrane-engineered EVs. METHODS: Platelet-mimetic EVs (P-EVs) were fabricated by fusing the membranes of EVs with platelet membranes by extrusion. A mouse model of myocardial ischemia reperfusion (MI/R) was established and injected with PBS, EVs, and P-EVs to evaluate their targeting ability and therapeutic angiogenesis efficacy. RESULTS: P-EVs inherited the adhesive proteins and natural targeting ability to injured vasculature of platelets and retained the pro-angiogenic potential of EVs. In the MI/R model, P-EVs preferentially accumulated in the injured endothelium of the ischemic hearts and enhanced the angiogenesis potency of EVs. CONCLUSIONS: This engineering strategy to modify pre-isolated EVs with platelet membranes by membrane fusion bestows EVs with the targeting ability of platelets and offers an exciting opportunity to design other targeted EVs fused with cell membranes from different sources for therapeutic angiogenesis.

Monocyte mimics improve mesenchymal stem cell-derived extracellular vesicle homing in a mouse MI/RI model.

Stem cell-derived extracellular vesicles (EVs) have been demonstrated to be effective in heart repair and regeneration post infarction. However, the poor homing efficiency and low yields of these therapeutics remain the major obstacles before they can be used in the clinic. To improve the delivery efficiency of EVs to ischemia-injured myocardium, we modified mesenchymal stem cell (MSC)-derived EVs with monocyte mimics through the method of membrane fusion. Monocyte mimic-bioinspired MSC-EVs (Mon-Exos) exhibited enhanced targeting efficiency to injured myocardium by mimicking the recruitment feature of monocytes after MI/RI, thus contributing to these exclusive adhesive molecules on monocyte mimics, particularly the Mac1/LFA1-ICAM-1 interaction. Through this strategy, Mon-Exos were shown to promote endothelial maturation during angiogenesis and modulate macrophage subpopulations after MI/RI, consistent with MSC-Exos biofunctions, and eventually improve therapeutic outcomes in cardiac function and pathohistology changes after treatments in a mouse MI/RI model. Ultimately, this strategy might provide us with a better way to assess the effects of stem cell EVs and offer additional techniques to help clinicians better manage regenerative therapeutics for ischemic heart diseases.

Platelet Membrane-Coated Nanoparticles Target Sclerotic Aortic Valves in ApoE-/- Mice by Multiple Binding Mechanisms Under Pathological Shear Stress.

BACKGROUND: Aortic valve disease is the most common valvular heart disease leading to valve replacement. The efficacy of pharmacological therapy for aortic valve disease is limited by the high mechanical stress at the aortic valves impairing the binding rate. We aimed to identify nanoparticle coating with entire platelet membranes to fully mimic their inherent multiple adhesive mechanisms and target the sclerotic aortic valve of apolipoprotein E-deficient (ApoE-/-) mice based on their multiple sites binding capacity under high shear stress. METHODS: Considering the potent interaction of platelet membrane glycoproteins with components present in sclerotic aortic valves, platelet membrane-coated nanoparticles (PNPs) were synthetized and the binding capacity under high shear stress was evaluated in vitro and in vivo. RESULTS: PNPs demonstrated effectively adhering to von Willebrand factor, collagen and fibrin under shear stresses in vitro. In an aortic valve disease model established in ApoE-/- mice, PNPs exhibited good targeting to sclerotic aortic valves by mimicking platelet multiple adhesive mechanisms. CONCLUSION: PNPs could provide a promising platform for the molecular diagnosis and targeting treatment of aortic valve disease.

MircroRNA-10b Promotes Human Embryonic Stem Cell-Derived Cardiomyocyte Proliferation via Novel Target Gene LATS1.

Adult mammalian cardiomyocytes (CMs) retain a limited proliferative ability, which is insufficient for the repair of CM loss in ischemic cardiac injury. Regulation of the Hippo signaling pathway to promote endogenous CM proliferation has emerged as a promising strategy for heart regeneration. Previous studies have shown that the microRNA cluster miR302-367 negatively regulates the Hippo pathway, promoting CM proliferation. In this study, we identified another microRNA, miR-10b, that regulates the Hippo pathway and promotes cell proliferation in human embryonic stem cell-derived CMs (hESC-CMs). We observed that miR-10b expression was enriched in the early stage of CMs, but its expression was reduced over time. Overexpression of miR-10b promoted CM proliferation, while knockdown of miR-10b suppressed CM proliferation. Moreover, miR-10b protected CMs against apoptosis. miR-10b functions, in part, by directly targeting LATS1, which is a major component of the Hippo pathway. Our study suggests that miR-10b has promising potential for heart regeneration.

Modification with CREKA Improves Cell Retention in a Rat Model of Myocardial Ischemia Reperfusion.

Poor cell homing limits the efficacy of cardiac cellular therapy. The homing peptide, cysteine-arginine-glutamic acid-lysine-alanine (CREKA), targets fibrin effectively which is involved in the repair process of tissue injury. Here, we assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots (2.6- and 2.3-fold, respectively). CREKA-MSCs showed 6.5-fold higher accumulation than unmodified MSCs in injured rat myocardium one day after administration, resulting in better structural preservation and functional recovery. Fibrin is, therefore, a novel target for enhancing homing of transplanted cells to injured myocardium, and the delivery system of fibrin-targeting is on behalf of a universalizable platform technology for regenerative medicine. Stem Cells 2019;37:663-676.