Targeted heart repair by Tß4-loaded cardiac-resident macrophage-derived extracellular vesicles modified with monocyte membranes.

Recent studies have demonstrated the critical role of cardiac-resident macrophages (cMacs) in the maintenance of physiological homeostasis. However, recruitment of circulating monocyte-derived macrophages decreases cMac levels post-myocardial infarction (MI). Transplanting cMacs is not an ideal option due to their low survival rates and the risk of immunological rejection. However, extracellular vesicle therapy has the potential to provide a feasible and safe alternative for cardiac repair. In this study, cell membrane-modified extracellular vesicles (MmEVs) were developed for heart repair by modifying cMac-derived extracellular vesicles (mEVs) with monocyte membranes, resulting in immune evasion and sequential targeted localization to damaged regions through expression of CD47 on MmEVs and strong affinity between monocyte membrane proteins and CCL2. Additionally, to fully exploit the potential clinical application of MmEVs and achieve a better curative effect, thymosin ß4 (Tß4) was loaded into the nanoparticles, resulting in Tß4-MmEVs. In vitro experiments indicated that both the MmEVs and Tß4-MmEVs promoted cardiomyocyte proliferation and endothelial cell migration. Animal experiments suggested that MI mice treated with MmEVs and Tß4-MmEVs exhibited reduced myocardial fibrosis and increased vascular density compared to the control group. Thus, we posit that these targeted nanoparticles hold significant potential for MI adjuvant therapy and may open new avenues for cardiac repair and regeneration. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) derived from bioactive parent cell sources involved in pathological and repair processes for cardiovascular disease have emerged as a compelling strategy for regenerative therapy. In this study, we constructed monocyte membrane-modified extracellular vesicles loaded with a drug (Tß4-MmEVs) for heart repair that exhibit extraordinary abilities of immune evasion and sequential localization to damaged regions owing to the presence of CD47 and the strong affinity between monocytes and damaged cardiomyocytes and endothelial cells. The bioactivities of Tß4-MmEVs on enhancing cardiomyocyte and endothelial cell proliferation were validated both in vitro and in vivo. Effective development and implementation of therapeutically membrane-modified nanoparticles from homologous origins can provide a reference for adjuvant therapy in clinical MI management.

Supramolecular Assemblies of Glycopeptides Enhance Gap Junction Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes via Inducing Spheroids Formation to Optimize Cardiac Repair.

Stem cell-based therapies have demonstrated significant potential for use in heart regeneration. An effective paradigm for heart repair in rodent and large animal models is the transplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Despite this, the functional and phenotypical immaturity of 2D-cultured hiPSC-CMs, particularly their low electrical integration, poses a caveat for clinical translation. In this study, a supramolecular assembly of a glycopeptide containing a cell adhesion motif-RGD, and saccharide-glucose (Bio-Gluc-RGD) is designed to enable the 3D spheroid formation of hiPSC-CMs, promoting cell-cell and cell-matrix interactions that occur during spontaneous morphogenesis. HiPSC-CMs in spheroids are prone to be phenotypically mature and developed robust gap junctions via activation of the integrin/ILK/p-AKT/Gata4 pathway. Monodispersed hiPSC-CMs encapsulated in the Bio-Gluc-RGD hydrogel are more likely to form aggregates and, therefore, survive in the infarcted myocardium of mice, accompanied by more robust gap junction formation in the transplanted cells, and hiPSC-CMs delivered with the hydrogels also displayed angiogenic effect and anti-apoptosis capacity in the peri-infarct area, enhancing their overall therapeutic efficacy in myocardial infarction. Collectively, the findings illustrate a novel concept for modulating hiPSC-CM maturation by spheroid induction, which has the potential for post-MI heart regeneration.

Compound Danshen Dripping Pills pretreatment protects the heart from ischemia/reperfusion injury by enhancing autophagic flux

Abstract Compound Danshen Dripping Pills (CDDPs) have been used in clinical treatment to protect the heart from ischemia/reperfusion (IR) injury for many years. However, the underlying mechanism implicated in the protective effects remains to be explored. Here, we determined the effects of CDDPs in Sprague-Dawley rats with the IR model. Cardiac function in vivo was assessed by echocardiography. Transmission electron microscopy, histological and immunohistochemical techniques, Western blotting and recombinant adeno-associated virus 9 transfection were used to illustrate the effects of CDDPs on IR and autophagy. Our results showed that pretreatment with CDDPs decreased the level of serum myocardial enzymes and infarct size in rats after IR. Apoptosis evaluation showed that CDDPs significantly ameliorated the cardiac apoptosis level after IR. Meanwhile, CDDPs pretreatment increased myocardial autophagic flux, with upregulation of LC3B, downregulation of p62, and increased autophagosomes and autolysosomes. Moreover, the autophagic flux inhibitor chloroquine could increase IR injury, while CDDPs could partially reverse the effects. Furthermore, our results showed that the activation of AMPK/mTOR was involved in the cardioprotective effect exerted by CDDPs. Herein, we suggest that CDDPs partially protect the heart from IR injury by enhancing autophagic flux through the activation of AMPK/mTOR.

Fabrication of Tß4-Exosome-releasing artificial stem cells for myocardial infarction therapy by improving coronary collateralization.

Currently, stem cell transplantations in cardiac repair are limited owing to disadvantages, such as immunological rejection and poor cell viability. Although direct injection of exosomes can have a curative effect similar to that of stem cell transplantation, high clearance hinders its application in clinical practice. Previous reports suggested that induction of coronary collateralization can be a desired method of adjunctive therapy for someone who had missed the optimal operation time to attenuate myocardial ischemia. In this study, to mimic the paracrine and biological activity of stem cells, we developed artificial stem cells that can continuously release Tß4-exosomes (Tß4-ASCs) by encapsulating specific exosomes within microspheres using microfluidics technology. The results show that Tß4-ASCs can greatly promote coronary collateralization in the periphery of the myocardial infarcted area, and its therapeutic effect is superior to that of directly injecting the exosomes. In addition, to better understand how it works, we demonstrated that the Tß4-ASC-derived exosomes can enhance the angiogenic capacity of coronary endothelial cells (CAECs) via the miR-17-5p/PHD3/Hif-1α pathway. In brief, as artificial stem cells, Tß4-ASCs can constantly release functional exosomes and stimulate the formation of collateral circulation after myocardial infarction, providing a feasible and alternative method for clinical revascularization.

Nkx2-3 induces autophagy inhibiting proliferation and migration of vascular smooth muscle cells via AMPK/mTOR signaling pathway.

Vascular remodeling and restenosis are common complications after percutaneous coronary intervention. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in intimal hyperplasia-induced vascular restenosis. NK2 Homeobox 3 (Nkx2-3), a critical member of Nkx family, is involved in tissue differentiation and organ development. However, the role of Nkx2-3 in VSMCs proliferation and migration remains unknown. In this study, we used carotid balloon injury model and platelet-derived growth factor-BB (PDGF)-treated VSMCs as in vivo and in vitro experimental models. EdU assay and CCK-8 assay were used to detect cell proliferation. Migration was measured by scratch test. Hematoxylin and eosin staining and immunohistochemistry staining were used to evaluate the intimal hyperplasia. The autophagy level was detected by fluorescent mRFP-GFP-LC3 in vitro and by transmission electron microscopy in vivo. It was shown that Nkx2-3 was upregulated both in balloon injured carotid arteries and PDGF-stimulated VSMCs. Adenovirus-mediated Nkx2-3 overexpression inhibited intimal hyperplasia after balloon injury, and suppressed VSMCs proliferation and migration induced by PDGF. Conversely, silencing of Nkx2-3 by small interfering RNA exaggerated proliferation and migration of VSMCs. Furthermore, we found that Nkx2-3 enhanced autophagy level, while the autophagy inhibitor 3-MA eliminated the inhibitory effect of Nkx2-3 on VSMCs proliferation and migration both in vivo and in vitro. Moreover, Nkx2-3 promoted autophagy in VSMCs by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. These results demonstrated for the first time that Nkx2-3 inhibited VSMCs proliferation and migration through AMPK/mTOR-mediated autophagy.

In situ activated mesenchymal stem cells (MSCs) by bioactive hydrogels for myocardial infarction treatment.

Stem-cell therapy has been proved as a promising strategy for myocardial infarction (MI) treatment. However, the therapeutic efficacy is mainly limited by the cellular activity of transplanted mesenchymal stem cells (MSCs). In this study, a novel bioglass (BG)/γ-polyglutamic acid (γ-PGA)/chitosan (CS) hydrogel was obtained by in situ adding BG to stimulate the imine bond formation. And the effect of the composite hydrogel on MI therapeutic efficacy was evaluated in a rat acute myocardial infarction (AMI) model in vivo and the possible mechanism of the BG/γ-PGA/CS hydrogel for the stimulation of the intercellular interaction between MSCs and cardiomyocytes (CMs) was explored by a MSC and CM co-culture experiment in vitro. The implantation of the MSC loaded BG/γ-PGA/CS composite hydrogel in the mice AMI model showed a significant improvement in the therapeutic efficacy with improved cardiac function, attenuation of heart remodeling, reduced cardiomyocyte apoptosis and accelerated vascularization. The in vitro cell experiments demonstrated that the BG/γ-PGA/CS hydrogel activated the intercellular interaction between MSCs and CMs, which resulted in reduced cell apoptosis and enhanced angiogenesis. Silicate based bioactive hydrogels activated MSCs and cell-cell interactions in cardiac tissue after AMI and significantly enhanced the efficacy, which suggests that this bioactive hydrogel based approach is an effective way to enhance stem-cell therapy.

Folic Acid Derived Hydrogel Enhances the Survival and Promotes Therapeutic Efficacy of iPS Cells for Acute Myocardial Infarction.

Stem cell therapy has obtained extensive consensus to be an effective method for post myocardial infarction (MI) intervention. Induced pluripotent stem (iPS) cells, which are able to differentiate into multiple cell types, have the potential to generate cardiovascular lineage cells for myocardial repair after ischemic damage, but their poor retention rate significantly hinders the therapeutic efficacy. In the present study, we developed a supramolecular hydrogel which is formed by the self-assembly of folic acid (FA)-modified peptide via a biocompatible method (glutathione reduction) and suitable for cell encapsulation and transplantation. The iPS cells labeled with CM-Dil were transplanted into the MI hearts of mice with or without FA hydrogel encapsulation. The results corroborated that the FA hydrogel significantly improved the retention and survival of iPS cells in MI hearts post injection, leading to augmentation of the therapeutic efficacy of iPS cells including better cardiac function and much less adverse heart remodeling, by subsequent differentiation toward cardiac cells and stimulation of neovascularization. This study reported a novel supramolecular hydrogel based on FA-peptides capable of improving the therapeutic capacity of iPS cells, which held big potential in the treatment of MI.

A Promising Therapeutic Target for Metabolic Diseases: Neuropeptide Y Receptors in Humans.

Human neuropeptide Y (hNPY) is one of the most widely expressed neurotransmitters in the human central and peripheral nervous systems. It consists of 36 highly conserved amino acid residues, and was first isolated from the porcine hypothalamus in 1982. While it is the most recently discovered member of the pancreatic polypeptide family (which includes neuropeptide Y, gut-derived hormone peptide YY, and pancreatic polypeptide), NPY is the most abundant peptide found in the mammalian brain. In order to exert particular functions, NPY needs to bind to the NPY receptor to activate specific signaling pathways. NPY receptors belong to the class A or rhodopsin-like G-protein coupled receptor (GPCR) family and signal via cell-surface receptors. By binding to GPCRs, NPY plays a crucial role in various biological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. Abnormal regulation of NPY is involved in the development of a wide range of diseases, including obesity, hypertension, atherosclerosis, epilepsy, metabolic disorders, and many cancers. Thus far, five receptors have been cloned from mammals (Y1, Y2, Y4, Y5, and y6), but only four of these (hY1, hY2, hY4, and hY5) are functional in humans. In this review, we summarize the structural characteristics of human NPY receptors and their role in metabolic diseases.

Treatment of Myocardial Infarction with Gene-modified Mesenchymal Stem Cells in a Small Molecular Hydrogel.

The effect of transplanted rat mesenchymal stem cells (MSCs) can be reduced by extracellular microenvironment in myocardial infarction (MI). We tested a novel small-molecular hydrogel (SMH) on whether it could provide a scaffold for hepatocyte growth factor (HGF)-modified MSCs and alleviate ventricular remodeling while preserving cardiac function after MI. Overexpression of HGF in MSCs increased Bcl-2 and reduced Bax and caspase-3 levels in response to hypoxia in vitro. Immunocytochemistry demonstrated that cardiac troponin (cTnT), desmin and connexin 43 expression were significantly enhanced in the 5-azacytidine (5-aza) with SMH group compared with the 5-aza only group in vitro and in vivo. Bioluminescent imaging indicated that retention and survival of transplanted cells was highest when MSCs transfected with adenovirus (ad-HGF) were injected with SMH. Heart function and structure improvement were confirmed by echocardiography and histology in the Ad-HGF-SMHs-MSCs group compared to other groups. Our study showed that: HGF alleviated cell apoptosis and promoted MSC growth. SMHs improved stem cell adhesion, survival and myocardial cell differentiation after MSC transplantation. SMHs combined with modified MSCs significantly decreased the scar area and improved cardiac function.

Lentiviral vector-mediated co-overexpression of VEGF and Bcl-2 improves mesenchymal stem cell survival and enhances paracrine effects in vitro.

Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for ischemic heart disease; however, the low survival rate of transplanted cells limits their therapeutic efficacy. The aim of this study was to investigate whether the dual genetic modification of vascular endothelial growth factor (VEGF) and B­cell lymphoma­2 (Bcl­2) confers a higher expression level of the target genes, better survival and a stronger paracrine effect in MSCs in an adverse environment than the modification of the individual genes. For this purpse, a lentiviral vector was constructed by using a self­cleaving T2A peptide sequence to link and achieve the co­overexpression of VEGF and Bcl­2. Rat MSCs were transfected to obtain cell lines that exhibited a stable overexpression. An in vitro model of oxygen glucose deprivation (OGD) was applied to mimic the ischemic microenvironment, and cell apoptosis, autophagy and the paracrine effects were then determined. Compared with the MSCs in which individual genes were modified and the control MSCs, the MSCs which were subjected to dual genetic modification had a higher expression level of the target genes, a more rapid proliferation, reduced apoptosis, decreased autophagy and an enhanced paracrine effect. Furthermore, the suppression of autophagy was found to contribute to the inhibition of apoptosis in this in vitro OGD model. On the whole, these data indicate that the co­overexpression of VEGF and Bcl­2 protects MSCs in an ischemic environment by inhibiting apoptosis, suppressing autophagy and enhancing the paracrine effects.