Layer-By-Layer Fabrication of Thicker and Larger Human Cardiac Muscle Patches for Cardiac Repair in Mice

The engineered myocardial tissues produced via most manufacturing techniques are typically just a few dozen micrometers thick, which is too thin for therapeutic applications in patients. Here, we used a modified layer-by-layer (LBL) fabrication protocol to generate thick human cardiac muscle patches (hCMPs) with thicknesses of ~3.75 mm. The LBL-hCMPs were composed of a layer of endothelial cells (ECs) sandwiched between two layers of cardiomyocytes (CMs): both cell populations were differentiated from the same human induced pluripotent stem cell line (hiPSCs) and suspended in a fibrin matrix, and the individual layers were sutured together, leaving channels that allowed the culture medium to access the internal cell layer. The LBL-hCMPs were cultured on a dynamic culture platform with electrical stimulation, and when compared to Control-hCMPs consisting of the same total number of hiPSC-ECs and -CMs suspended in a single layer of fibrin, hiPSC-CMs in the LBL-hCMPs were qualitatively more mature with significantly longer sarcomeres and expressed significantly higher levels of mRNA transcripts for proteins that participate in cardiomyocyte contractile activity and calcium handing. Apoptotic cells were also less common in LBL- than in Control-hCMPs. The thickness of fabricated LBL-hCMP gradually decreased to 0.8 mm by day 28 in dynamic culture. When the hCMP constructs were compared in a mouse model of myocardial infarction, the LBL-hCMPs were associated with significantly better measurements of engraftment, cardiac function, infarct size, hypertrophy, and vascularity. Collectively these observations indicate that our modified LBL fabrication protocol produced thicker hCMPs with no decline in cell viability, and that LBL-hCMPs were more potent than Control-hCMPs for promoting myocardial repair in mice.

Therapeutic Applications of Extracellular Vesicles for Myocardial Repair

Cardiovascular disease is the leading cause of human death worldwide. Drug thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting and other methods are used to restore blood perfusion for coronary artery stenosis and blockage. The treatments listed prolong lifespan, however, rate of mortality ultimately remains the same. This is due to the irreversible damage sustained by myocardium, in which millions of heart cells are lost during myocardial infarction. The lack of pragmatic methods of myocardial restoration remains the greatest challenge for effective treatment. Exosomes are small extracellular vesicles (EVs) actively secreted by all cell types that act as effective transmitters of biological signals which contribute to both reparative and pathological processes within the heart. Exosomes have become the focus of many researchers as a novel drug delivery system due to the advantages of low toxicity, little immunogenicity and good permeability. In this review, we discuss the progress and challenges of EVs in myocardial repair, and review the recent development of extracellular vesicle-loading systems based on their unique nanostructures and physiological functions, as well as the application of engineering modifications in the diagnosis and treatment of myocardial repair.

Regenerating Damaged Myocardium: A Review of Stem-Cell Therapies for Heart Failure

Cardiovascular disease (CVD) is one of the contributing factors to more than one-third of human mortality and the leading cause of death worldwide. The death of cardiac myocyte is a fundamental pathological process in cardiac pathologies caused by various heart diseases, including myocardial infarction. Thus, strategies for replacing fibrotic tissue in the infarcted region with functional myocardium have long been a goal of cardiovascular research. This review begins by briefly discussing a variety of somatic stem- and progenitor-cell populations that were frequently studied in early investigations of regenerative myocardial therapy and then focuses primarily on pluripotent stem cells (PSCs), especially induced-pluripotent stem cells (iPSCs), which have emerged as perhaps the most promising source of cardiomyocytes for both therapeutic applications and drug testing. We also describe attempts to generate cardiomyocytes directly from cardiac fibroblasts (i.e., transdifferentiation), which, if successful, may enable the pool of endogenous cardiac fibroblasts to be used as an in-situ source of cardiomyocytes for myocardial repair.

Regulation of Transplanted Cell Homing by FGF1 and PDGFB after Doxorubicin Myocardial Injury

There is no effective treatment for the total recovery of myocardial injury caused by an anticancer drug, doxorubicin (Dox). In this study, using a Dox-induced cardiac injury model, we compared the cardioprotective effects of ventricular cells harvested from 11.5-day old embryonic mice (E11.5) with those from E14.5 embryos. Our results indicate that tail-vein-infused E11.5 ventricular cells are more efficient at homing into the injured adult myocardium, and are more angiogenic, than E14.5 ventricular cells. In addition, E11.5 cells were shown to mitigate the cardiomyopathic effects of Dox. In vitro, E11.5 ventricular cells were more migratory than E14.5 cells, and RT-qPCR analysis revealed that they express significantly higher levels of cytokine receptors Fgfr1, Fgfr2, Pdgfra, Pdgfrb and Kit. Remarkably, mRNA levels for Fgf1, Fgf2, Pdgfa and Pdgfb were also found to be elevated in the Dox-injured adult heart, as were the FGF1 and PDGFB protein levels. Addition of exogenous FGF1 or PDGFB was able to enhance E11.5 ventricular cell migration in vitro, and, whereas their neutralizing antibodies decreased cell migration. These results indicate that therapies raising the levels of FGF1 and PDGFB receptors in donor cells and or corresponding ligands in an injured heart could improve the efficacy of cell-based interventions for myocardial repair.

Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

Background The neonatal heart maintains its entire regeneration capacity within days after birth. Using quantitative phosphoproteomics technology, we identified that SGK3 (serine/threonine‐protein kinase 3) in the neonatal heart is highly expressed and activated after myocardial infarction. This study aimed to uncover the function and related mechanisms of SGK3 on cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. Methods and Results The effect of SGK3 on proliferation and oxygen glucose deprivation/reoxygenation– induced apoptosis in isolated cardiomyocytes was evaluated using cardiomyocyte‐specific SGK3 overexpression or knockdown adenovirus5 vector. In vivo, gain‐ and loss‐of‐function experiments using cardiomyocyte‐specific adeno‐associated virus 9 were performed to determine the effect of SGK3 in cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. In vitro, overexpression of SGK3 enhanced, whereas knockdown of SGK3 decreased, the cardiomyocyte proliferation ratio. In vivo, inhibiting the expression of SGK3 shortened the time window of cardiac regeneration after apical resection in neonatal mice, and overexpression of SGK3 significantly promoted myocardial repair and cardiac function recovery after ischemia/reperfusion injury in adult mice. Mechanistically, SGK3 promoted cardiomyocyte regeneration and myocardial repair after cardiac injury by inhibiting GSK‐3β (glycogen synthase kinase‐3β) activity and upregulating β‐catenin expression. SGK3 also upregulated the expression of cell cycle promoting genes G1/S‐specific cyclin‐D1, c‐myc (cellular‐myelocytomatosis viral oncogene), and cdc20 (cell division cycle 20), but downregulated the expression of cell cycle negative regulators cyclin kinase inhibitor P 21 and cyclin kinase inhibitor P 27. Conclusions Our study reveals a key role of SGK3 on cardiac repair after apical resection or ischemia/reperfusion injury, which may reopen a novel therapeutic option for myocardial infarction.

Endothelial regeneration in repair from vascular injury and stem cell biology

Professor Teruo Inoue and his collaborators are exploring repair from vascular and myocardial injury in the context of stem cell biology in work that is set to make waves in regenerative medicine. This research involves endothelial progenitor cells (EPCs) for vascular repair, adipose-derived regenerative cells (ADRCs) for angiogenesis and multilineage differentiating stress enduring cell (Muse) cells for myocardial repair. Inoue and his collaborators are also investigating the 'wound repair priming' phenomenon with a view to overcoming the challenge of the inconsistent capacities of angiogenesis due to individual differences in cell quality. The researchers found that the ADRCs (also called adipose-derived stromal fractions: SVFs) obtained from subcutaneous fat after manipulation caused by surgical injury as well as ischaemia showed higher angiogenetic ability. The researchers plan to pharmacologically reproduce wound repair priming in order to facilitate more consistent cell therapy using ADRCs. The team is also exploring other promising functional analysis methods for stem cells, including comprehensive gene analysis using single cell RNA sequence (scRNA seq), which the researchers plan to apply to Muse cells. In addition to Muse cell research targeting myocardial repair, the team will also conduct Muse cell vascular research as they believe that Muse cell treatment holds great promise for the repair of injured-vessel sites. Ultimately, Inoue and his collaborators hope their work will significantly impact medical research and clinical medicine.

Targeted delivery controlled release of hepatic growth factor and insulin-like growth factor-1 improves left ventricular repair in a porcine model of myocardial ischemia reperfusion injury

Introduction Nanomedicine offers great potential for treatment of cardiovascular disease. We tested whether direct intramyocardial (IM) injection of pro-angiogenic hepatocyte growth factor (HGF) and pro-myogenic insulin-like growth factor (IGF-1) encapsulated in Alginate-Sulfate nanoparticles (AlgS-NP) enhances myocardial retention, controlled release and improves myocardial repair in a porcine ischemia-reperfusion model. Methods Bioactivity of HGF/IGF, released from AlgS-NP, was determined by cell proliferation assays in vitro. Myocardial infarction (MI) was induced by 75min balloon occlusion of the mid-LAD followed by reperfusion. After 1w, pigs (n=12) with marked LV dysfunction (EF<45%) were randomized to fusion imaging-guided IM injections of 8 mg Cy5-labelled AlgS-NP loaded with 200μg HGF and 200μg IGF-1 (GF) or with phosphate-buffered saline (CON) using the MYOSTAR injection catheter. AlgS-NP retention after IM or intracoronary (IC) injection was determined by measuring Cy5 plasma levels. At 8w, treatment effect was evaluated using in vivo magnetic resonance imaging and coronary physiological measurements, and via post-mortem analysis of myocardial fibrosis and cardiomyocyte circumference. Results We confirmed the bioactivity of the AlgS-NP-released GF in C2C12 and HUVEC cell proliferation assays after 72h culture, being similar to the free GF (Fig. A). AlgS-NP retention was tested in a pig model, 1w after MI. Ejection fraction (EF) was 37±5% (range 27–45%) and infarct size (IS)/LVmass 24±6% (range 19–38%). AlgS-NP retention was better after IM delivery than after IC infusion with plasma Cy5 levels at 30 min after treatment indicating 5% systemic leakage for IM vs. 20% for IC. After 8w, IS/LVmass decreased 8% in GF-treated pigs vs. 3% in CON (P=0.03, Fig. B) and was associated with preserved myocardial blood flow during hyperemia in the infarct (P=0.036) and peri-infarct (PI) zones (P=0.008), increased coronary flow reserve (P=0.05) and decreased index of microcirculatory resistance (P=0.02). LVEF significantly increased in GF-treated pigs (+6±2% vs. −1±1% in CON, P=0.02, Fig. C), and was accompanied by significantly reduced fibrosis (P=0.01) and increased hypertrophy of cardiomyocyte (P=0.03) in the PI zone. Conclusions IM injection of AlgS-NP-encapsulated HGF and IGF-1 to the ischemic myocardium significantly improves LV repair, and offers the prospect of innovative treatment for patients with refractory ischemic heart disease. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EuroNanoMed II Figure A Figure B and C

Smooth muscle cell specific ablation of CXCL12 downregulates endothelial CXCR7 leading to defective coronary arteries and cardiac hypertrophy

Aims The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). Methods and results SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Conclusion Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FWF-Austria