Intramyocardial Injection Research Articles

Cardiopulmonary progenitors facilitate cardiac repair via exosomal transfer of miR-27b-3p targeting the SIK1-CREB1 axis.

Ischemic heart disease, especially myocardial infarction (MI), is one of the leading causes of death worldwide, and desperately needs effective treatments, such as cell therapy. Cardiopulmonary progenitors (CPPs) are stem cells for both heart and lung, but their repairing role in damaged heart is still unknown. Here, we obtained CPPs from E9.5 mouse embryos, maintained their stemness while expanding, and identified their characteristics by scRNA-seq, flow cytometry, quantitative reverse transcription-polymerase chain reaction, and differentiation assays. Moreover, we employed mouse MI model to investigate whether CPPs could repair the injured heart. Our data identified that CPPs exhibit hybrid fibroblastic, endothelial, and mesenchymal state, and they could differentiate into cell lineages within the cardiopulmonary system. Moreover, intramyocardial injection of CPPs improves cardiac function through CPPs exosomes (CPPs-Exo) by promotion of cardiomyocytic proliferation and vascularization. To uncover the underlying mechanism, we used miRNA-seq, bulk RNA-seq, and bioinformatic approaches, and found the highly expressed miR-27b-3p in CPPs-Exo and its target gene Sik1, which can influence the transcriptional activity of CREB1. Therefore, we postulate that CPPs facilitate cardiac repair partially through the SIK1-CREB1 axis via exosomal miR-27b-3p. Our study offers a novel insight into the role of CPPs-Exo in heart repair and highlights the potential of CPPs-Exo as a promising therapeutic strategy for MI.

Comparative effectiveness of myocardial patches and intramyocardial injections in treating myocardial infarction with a MitoQ/hydrogel system.

In cardiac tissue engineering, myocardial surface patches and hydrogel intramyocardial injections represent the two primary hydrogel-based strategies for myocardial infarction (MI) treatment. However, the comparative effectiveness of these two treatments remains uncertain. Therefore, this study aimed to compare the effects of the two treatment modalities by designing a simple and reproducible hydrogel cross-linked with γ-PGA and 4-arm-PEG-SG. To improve mitochondrial damage in cardiomyocytes (CMs) during early MI, we incorporated the mitochondria-targeting antioxidant MitoQ into the hydrogel network. The hydrogel exhibited excellent biodegradability, biocompatibility, adhesion, and injectability in vitro. The hydrogel was utilized for rat MI treatment through both patch adhesion and intramyocardial injections. In vivo results demonstrated that the slow release of MitoQ peptide from the hydrogel hindered ROS production in CM, alleviated mitochondrial damage, and enhanced CM activity within 7 days, effectively inhibiting MI progression. Both hydrogel intramyocardial injections and patches exhibited positive therapeutic effects, with intramyocardial injections demonstrating superior efficacy in terms of cardiac function and structure in equivalent treatment cycles. In conclusion, we developed a MitoQ/hydrogel system that is easily prepared and can serve as both a myocardial patch and an intramyocardial injection for MI treatment, showing significant potential for clinical applications.

Inactivation of the NLRP3 inflammasome mediates exosome-based prevention of atrial fibrillation.

Rationale: Extracellular vesicles (EVs) from human explant-derived cells injected directly into the atria wall muscle at the time of open chest surgery reduce atrial fibrosis, atrial inflammation, and atrial fibrillation (AF) in a rat model of sterile pericarditis. Albeit a promising solution to prevent postoperative AF, the mechanism(s) underlying this effect are unknown and it is not clear if this benefit is dependent on EV dose. Methods: To determine the dose-efficacy relationship of EVs from human explant-derived cells in a rat model of sterile pericarditis. Increasing doses of EVs (106, 107, 108 or 109) or vehicle control were injected into the atria of middle-age male Sprague-Dawley rats at the time of talc application. A sham control group was included to demonstrate background inducibility. Three days after surgery, all rats underwent invasive electrophysiological testing prior to sacrifice. Results: Pericarditis increased the likelihood of inducing AF (p<0.05 vs. sham). All doses decreased the probability of inducing AF with maximal effects seen after treatment with the highest dose (109, p<0.05 vs. vehicle). Pericarditis increased atrial fibrosis while EV treatment limited the effect of pericarditis on atrial fibrosis with maximal effects seen after treatment with 108 or 109 EVs. Increasing EV dose was associated with progressive decreases in pro-inflammatory cytokine content, inflammatory cell infiltration, and oxidative stress. EVs decreased NLRP3 (NACHT, LRR, and PYD domains-containing protein-3) inflammasome activation though a direct effect on resident atrial fibroblasts and macrophages. This suppressive effect was exclusive to EVs produced by heart-derived cells as application of EVs from bone marrow or umbilical cords did not alter NLRP3 activity. Conclusions: Intramyocardial injection of incremental doses of EVs at the time of open chest surgery led to progressive reductions in atrial fibrosis and inflammatory markers. These effects combined to render atria resistant to the pro-arrhythmic effects of pericarditis which is mechanistically related to suppression of the NLRP3 inflammasome.

Astragaloside IV-induced BMSC exosomes promote neovascularization and protect cardiac function in myocardial infarction mice via the miR-411/HIF-1α axis

This study focused on investigating the mechanism of the astragaloside IV-induced bone marrow mesenchymal stem cell exosome (AS-IV-MSC-exo)/microRNA(miR)-411/HIF-1α axis in affecting vascular neovascularization and protecting cardiac function in myocardial infarction (MI) mice. Exosomes (MSC-exo and AS-IV-MSC-exo) were separated by differential centrifugation and then characterized. MI mouse models were established by left anterior descending coronary artery ligation. Echocardiography was used to evaluate cardiac function. HE staining and Masson staining were performed to observe myocardial histopathology. Capillary density in the myocardium via immunohistochemistry and quantified the expression of vascular endothelial growth factor (VEGF) via RT-qPCR. The expression of miR-411 and HIF-1α was tested by RT-qPCR and western blot and the targeting relationship of miR-411 and HIF-1α was verified by bioinformatics website and dual luciferase reporter gene assay. Exosomes with lipid bi-layer membrane structure, expressing exosomal surface marker proteins, and being taken up by cardiomyocytes could be successfully isolated utilizing ultracentrifugation. Intramyocardial injection of MSC-exo could restore cardiac function, decrease myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice; the effect of AS-IV-MSC-exo was more significant. The ability of AS-IV-MSC-exo to restore cardiac function, lower myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice was diminished when miR-411 expression in AS-IV-MSC-exo was reduced. Mechanistically, miR-411 was found to target and inhibit HIF-1α expression. Overexpression of HIF-1α impaired the impact of AS-IV-MSC-exo on improving cardiac function and promoting neovascularization in MI mice. AS-IV-MSC-exo improves cardiac function and promoted neovascularization via the miR-411/HIF-1α axis, thereby ameliorating MI.

Lack of cardiac benefit after intramyocardial or intravenous injection of mesenchymal stem cell-derived extracellular vesicles supports the need for optimized cardiac delivery.

To determine the differences in improvement in cardiac function by intramyocardial (IM) vs. intravenous (IV) injection of human bone mesenchymal stem cell-derived extracellular vesicles (HBMSC-EV) after acute MI. FVB mice underwent acute MI via left anterior descending coronary artery ligation and subsequent injection of: (1) IM saline control; (2) IM HBMSC-EV; (3) IV saline control; and (4) IV HBMSC-EV. Cardiac function was evaluated with weekly postoperative echocardiography. On postoperative day 28, the mice were euthanized, and the heart, lungs, liver, spleen, and kidneys were harvested. Given previous studies showing HBMSC-EV hepatic uptake after IV injection, the liver was evaluated for changes in inflammation, fibrosis, and proliferation. On postoperative day 28, there were no significant differences in left ventricular ejection fraction (P = 0.6151), fractional shortening (P = 0.1135), or anterior border zone fibrosis (P = 0.6333) in any of the experimental groups. Interestingly, there was a strong trend demonstrating improvement in infarct size on fibrosis staining, which did not reach significance (P = 0.05620). There were no differences in hepatic inflammation, fibrosis, and proliferation. Although there was a trend in the improvement in infarct size, a single-dose administration of neither IM nor IV injection of HBMSC-EV resulted in significant improvement in post-MI cardiac function. A major limitation of this study is the lack of trials determining the optimal dose of HBMSC-EV needed in this model. However, the current study demonstrates that future studies are required to either optimize administration or bioengineer HBMSC-EV with cardiac-homing properties.

PD-L1 and AKT Overexpressing Adipose-Derived Mesenchymal Stem Cells Enhance Myocardial Protection by Upregulating CD25+ T Cells in Acute Myocardial Infarction Rat Model.

This study explores the synergistic impact of Programmed Death Ligand 1 (PD-L1) and Protein Kinase B (Akt) overexpression in adipose-derived mesenchymal stem cells (AdMSCs) for ameliorating cardiac dysfunction after myocardial infarction (MI). Post-MI adult Wistar rats were allocated into four groups: sham, MI, ADMSC treatment, and ADMSCs overexpressed with PD-L1 and Akt (AdMSC-PDL1-Akt) treatment. MI was induced via left anterior descending coronary artery ligation, followed by intramyocardial AdMSC injections. Over four weeks, cardiac functionality and structural integrity were assessed using pressure-volume analysis, infarct size measurement, and immunohistochemistry. AdMSC-PDL1-Akt exhibited enhanced resistance to reactive oxygen species (ROS) in vitro and ameliorated MI-induced contractile dysfunction in vivo by improving the end-systolic pressure-volume relationship and preload-recruitable stroke work, together with attenuating infarct size. Molecular analyses revealed substantial mitigation in caspase3 and nuclear factor-κB upregulation in MI hearts within the AdMSC-PDL1-Akt group. Mechanistically, AdMSC-PDL1-Akt fostered the differentiation of normal T cells into CD25+ regulatory T cells in vitro, aligning with in vivo upregulation of CD25 in AdMSC-PDL1-Akt-treated rats. Collectively, PD-L1 and Akt overexpression in AdMSCs bolsters resistance to ROS-mediated apoptosis in vitro and enhances myocardial protective efficacy against MI-induced dysfunction, potentially via T-cell modulation, underscoring a promising therapeutic strategy for myocardial ischemic injuries.

Hydrogel‐Based Delivery of antimiR‐195 Improves Cardiac Efficacy after Ischemic Injury

AbstractMicroRNAs (miRs) are potent regulators of biology and disease. The miR‐15 family is shown to regulate cardiomyocyte proliferation and antimiR‐based inhibition induces a cardioprotective effect after myocardial infarction in mice. However, systemic delivery of antimiRs leads to accumulation in kidneys and liver, with relatively poor cardiac exposure. Injectable hydrogels are proposed to serve as sustained‐release drug delivery depots and can potentially be used to improve cardiac efficacy of antimiR therapeutics. Here, the effect of a hydrogel‐formulated antimiR‐195 after myocardial infarction in mice is studied. For this, an injectable, pH‐switchable supramolecular hydrogel based on poly(ethylene glycol) (PEG) functionalized with hydrogen bonding ureido‐pyrimidinone (UPy) units is used. Intracardiac injections under baseline conditions of this UPy–PEG hydrogelator induce a transient inflammatory response that is no longer present 7 days postinjection. In vitro experiments show that antimiR‐195 is released from the gel, and induces microRNA inhibition leading to downstream cardiomyocyte proliferation. In vivo, intramyocardial delivery of antimiR‐195 in UPy–PEG enhances cardiac target derepression compared to phosphate‐buffered‐saline‐dissolved antimiR‐195, despite a low cardiac retention. After ischemic injury, this translates into a greater therapeutic effect by increasing both target derepression and cardiomyocyte proliferation. Intramyocardial injection of UPy–PEG‐formulated antimiR‐195 is sufficient to improve cardiac efficacy of antimiR‐195.

Chemokine CCL2 promotes cardiac regeneration and repair in myocardial infarction mice via activation of the JNK/STAT3 axis.

Stimulation of adult cardiomyocyte proliferation is a promising strategy for treating myocardial infarction (MI). Earlier studies have shown increased CCL2 levels in plasma and cardiac tissue both in MI patients and mouse models. In present study we investigated the role of CCL2 in cardiac regeneration and the underlying mechanisms. MI was induced in adult mice by permanent ligation of the left anterior descending artery, we showed that the serum and cardiac CCL2 levels were significantly increased in MI mice. Intramyocardial injection of recombinant CCL2 (rCCL2, 1 μg) immediately after the surgery significantly promoted cardiomyocyte proliferation, improved survival rate and cardiac function, and diminished scar sizes in post-MI mice. Alongside these beneficial effects, we observed an increased angiogenesis and decreased cardiomyocyte apoptosis in post-MI mice. Conversely, treatment with a selective CCL2 synthesis inhibitor Bindarit (30 μM) suppressed both CCL2 expression and cardiomyocyte proliferation in P1 neonatal rat ventricle myocytes (NRVMs). We demonstrated in NRVMs that the CCL2 stimulated cardiomyocyte proliferation through STAT3 signaling: treatment with rCCL2 (100 ng/mL) significantly increased the phosphorylation levels of STAT3, whereas a STAT3 phosphorylation inhibitor Stattic (30 μM) suppressed rCCL2-induced cardiomyocyte proliferation. In conclusion, this study suggests that CCL2 promotes cardiac regeneration via activation of STAT3 signaling, underscoring its potential as a therapeutic agent for managing MI and associated heart failure.

TBC1 domain family member 25 protects against myocardial apoptosis and the proinflammatory response triggered by ischemia-reperfusion injury through suppression of the TAK1-JNK/p38 MAPK signaling cascade.

TBC1 domain family member 25 (TBC1D25) is a crucial mediator of signal transduction involved in the development of several diseases. Particularly, a cardioprotective role of TBC1D25 has been raised due to its antagonistic action on cardiac hypertrophy. However, whether TBC1D25 protects the myocardium from ischemia-reperfusion injury has not been reported. This work aimed to determine the role of TBC1D25 in myocardial ischemia-reperfusion (MIR) injury and to explore the potential mechanisms involved. Marked decreases in TBC1D25 levels occurred in cardiomyocytes suffering hypoxia/reoxygenation (H/R) injury in vitro and myocardium tissues of rats with MIR injury in vivo. Cardiomyocytes overexpressing TBC1D25 were protected from apoptosis and inflammation triggered by H/R, whereas TBC1D25-deficient cardiomyocytes were more sensitive to H/R injury. Intramyocardial injection of recombinant adenovirus expressing TBC1D25 into rats reduced infarct size and cardiac injury triggered by MIR injury accompanied by decreased myocardial apoptosis and inflammation. A subsequent mechanistic investigation revealed that the signaling cascade of transforming growth factor-β-activated kinase 1 (TAK1)-c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) activated under H/R or MIR conditions was markedly restrained by TBC1D25 overexpression. Moreover, TAK1 blockade remarkably reversed the TBC1D25 deficiency-induced aggravating effect on H/R injury. The work concludes that TBC1D25 protects against MIR injury through action on the TAK1-JNK/p38 MAPK signaling cascade. This work suggests TBC1D25 as a potential therapeutic target for MIR injury.

Investigating the effect of intramyocardial injection of DNAzyme-containing nisosome of NF-κB gene on the level of cardiac tissue NF-κB protein expression in cardiac ischemia-reperfusion of male rats

Investigating the effect of intramyocardial injection of DNAzyme-containing nisosome of NF-κB gene on the level of cardiac tissue NF-κB protein expression in cardiac ischemia-reperfusion of male rats

Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.

Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

Extracellular vesicles derived from different tissues attenuate cardiac dysfunction in murine MI models

BackgroundExtracellular vesicles (EVs) derived from various cell sources exert cardioprotective effects during cardiac ischemic injury. Our previous study confirmed that EVs derived from ischemic-reperfusion injured heart tissue aggravated cardiac inflammation and dysfunction. However, the role of EVs derived from normal cardiac tissue in myocardial ischemic injury remains elusive.ResultsIn the present study, normal heart-derived EVs (cEVs) and kidney-derived EVs (nEVs) were isolated and intramyocardially injected into mice after myocardial infarction (MI). We demonstrated that administration of both cEVs and nEVs significantly improved cardiac function, reduced the scar size, and alleviated inflammatory infiltration into the heart. In addition, cardiomyocyte apoptosis was inhibited, whereas angiogenesis was enhanced in the hearts receiving cEVs or nEVs treatment. Moreover, intramyocardial injection of cEVs displayed much better cardiac protective efficacy than nEVs in murine MI models. RNA-seq and protein-protein interaction (PPI) network analysis revealed the protective mRNA clusters in both cEVs and nEVs. These mRNAs were involved in multiple signaling pathways, which may synergistically orchestrate to prevent the heart from further damage post MI.ConclusionsCollectively, our results indicated that EVs derived from normal heart tissue may represent a promising strategy for cardiac protection in ischemic heart diseases.

Development of a potency assay for CD34+ cell-based therapy

We have previously shown that intracardiac delivery of autologous CD34+ cells after acute myocardial infarction (AMI) is safe and leads to long term improvement. We are now conducting a multicenter, randomized, controlled Phase I/IIb study in post-AMI to investigate the safety and efficacy of intramyocardial injection of expanded autologous CD34+ cells (ProtheraCytes) (NCT02669810). Here, we conducted a series of in vitro studies characterizing the growth factor secretion, exosome secretion, gene expression, cell surface markers, differentiation potential, and angiogenic potential of ProtheraCytes clinical batches to develop a potency assay. We show that ProtheraCytes secrete vascular endothelial growth factor (VEGF) and its concentration is significantly correlated with the number of CD34+ cells obtained after expansion. ProtheraCytes also secrete exosomes containing proangiogenic miRNAs (126, 130a, 378, 26a), antiapoptotic miRNAs (21 and 146a), antifibrotic miRNAs (133a, 24, 29b, 132), and miRNAs promoting myocardial regeneration (199a and 590). We also show that ProtheraCytes have in vitro angiogenic activity, express surface markers of endothelial progenitor cells, and can differentiate in vitro into endothelial cells. After the in vitro characterization of multiple ProtheraCytes clinical batches, we established that measuring the concentration of VEGF provided the most practical, reliable, and consistent potency assay.

Cardiotoxicity in light chain amyloidosis: Insights from the first murine model with intramyocardial injection of patient-derived amyloidogenic light chains

Abstract Background Light chain amyloidosis (AL) is a plasma cell dyscrasia in which cardiac involvement determines the prognosis. Cardiotoxicity of light chains (LCs) complicates the management of heart failure in AL and has not been addressed in the clinical praxis. We have previously genetically identified and biotechnologically produced full-length LCs from patients with AL and cardiac involvement and isotype LCs (WT) from healthy volunteers. We characterized the LCs’ toxicity on primary adult ventricular murine cardiomyocytes (CMs) and our investigation led to the selection of three cardiotoxic and amyloidogenic patient-derived LCs (one from the kappa and two from the lambda family) for further characterization. Purpose We aimed to 1) generate a murine model that recapitulates LCs’ cardiotoxicity in vivo, 2) characterize the mechanism of cardiotoxicity and 3) identify cardioprotective therapies. Methods C57BL6 mice were randomized into 6 groups as follows: 1) PBS (vehicle), 2) WT_kappa, 3) AL_kappa, 4) WT_lambda, 5) AL_lambda1 and 6) AL_lambda2. The animals received the LCs (1 mg/mouse) or vehicle via intramyocardial injection (IMI). Echocardiography evaluation and blood sampling was performed weekly. The animals were sacrificed on the 4th week post IMI for histology, electron microscopy and investigation of the cardiotoxicity mechanism. Results On the 4th week, the AL-lambda LCs significantly reduced the fractional shortening and ejection fraction compared to the vehicle and isotype control groups. The AL_lambda2 also increased mortality of the mice. The AL-kappa did not alter the systolic function of the mice. The circulating lactate dehydrogenase was significantly increased on the 2nd and 4th week in the AL-LCs treated mice indicative of the LCs’ toxicity. The IMI of AL_kappa led to signs of apoptosis including the shrinkage of cardiomyocytes and increased condensation of nuclear chromatin to the nuclear membranes. The IMI of AL_lambda LCs led to increased cardiomyocyte size indicative of hypertrophy, interstitial fibrosis, and increased number of autophagosomes. None of the animals bared amyloid deposits in the heart. The AL-LCs induced different molecular responses in vivo and confirmed the histology findings. The AL-kappa increased apoptosis via the CHOP-Bax pathway, and the ΑL-lambda increased Beclin-1 mediated autophagy without inducing apoptosis. We identified that the common upstream mediator of AL-LCs cardiotoxicity in vivo is the overexpression of the endoplasmic reticulum stress (ERS) markers, IRE-1a and Bip. In vitro, we confirmed that the AL-LCs induce cell death in CMs through ERS. Treatment with the IRE-1a inhibitor STF-083010 and the molecular chaperone tauroursodeoxycholic acid restored the LCs’ induced cardiotoxicity. Conclusions The IMI of AL-LCs stands as a method for the investigation of the LCs cardiotoxicity in vivo in the absence of amyloid deposits via which we identified ERS as a target to alleviate AL-LCs toxicity.

Cardioprotective potential of exosomes derived from blood outgrowth endothelial cells in ischemic cardiomyopathy

Abstract Introduction Blood outgrowth endothelial cell (BOEC)-derived exosomes can effectively induce vascular network formation in vitro and mediate therapeutic neovascularization in a skin wound healing model. We hypothesized that BOEC-derived exosomes can enhance myocardial repair in mice after ischemia-reperfusion (I/R) injury. Methods BOECs were isolated and culture-expanded from peripheral blood of patients with severe ischemic cardiomyopathy (ICMP) in normoxic and hypoxic (1% O2) conditions. Exosomes were isolated from the medium by differential ultracentrifugation, and their size, number, and surface markers were determined by nano tracking analysis (NTA) and immunoblot analysis. Myocardial infarction (MI) was induced via transient LAD ligation for 60 min followed by reperfusion. BOEC-derived exosomes and were resuspended in small fractions (120x10^8 particles per 10 µl) 2.5 µl 4 aliquots were injected under direct vision into the epicardial wall of the peri-infarct region. One month later, left ventricular (LV) structure and function were analyzed using a 7T Bruckner MRI with commercial software Segment in mice that received exosome injections or placebo injections (n=10 and n=8, respectively). Results Quantification of exosomes by NTA showed higher concentrations in the medium of BOECs cultured in hypoxia. Immunoblot analysis confirmed robust expression of exosome markers TSG101 and Flotilin-1. One month after intramyocardial exosome injections, heart rate, LV volumes or LV ejection fraction were similar between mice receiving myocardial exosomes injections (n=10) in the border zone as compared to those receiving placebo (n=8). However, in vivo exosome transfer mediated favorable structural remodeling after MI with lower LV mass indices (p=0.01) and better preserved regional wall thickness in all 4 segments receiving exosomes aliquots (p&amp;lt;0.05) (Figure). Conclusions Our results suggest that BOEC-derived exosomes have favorable post-infarction myocardial structural remodeling. Exosomes may constitute an attractive cell-free therapeutic modality.Figure

Transcatheter endomyocardial alginate-hydrogel injection for treatment of heart failure

Abstract Background and Aim Intramyocardial injection of alginate-hydrogel, an emerging and promising strategy for patients with advanced heart failure (HF), is limited by the paucity of commercially available intramyocardial delivery methods. This study investigates the feasibility and safety of a novel X-DROP catheter injection system (Figure 1) for delivery of the alginate-hydrogel to pre-defined target areas in the treatment of HF with reduced ejection fraction. Methods and results Prior to the First-in-man, we demonstrated that transcatheter endo-myocardial alginate-hydrogel implantation (TEAi) was successfully and precisely performed with the X-DROP catheter system under TEE and fluoroscopy guidance in a healthy porcine model (Figure 2). Acute animal results (n = 15) showed no mortality, perforation, and sustained ventricular arrhythmias. Gross examination revealed the retention of the alginate-hydrogel in the pre-defined endocardial area at six months post-procedure (n = 4, Figure 2). The histological assessment showed that alginate-hydrogel was encapsulated by multiple layers of fibrosis without causing damage to the nearby myocardium. Expanding from the animal result, we conducted a first-in-human trial to evaluate the TEAi in patients with heart failure. Ten patients with NYHA class III-IV were enrolled, and all patients were successfully treated with TEAi without the device or procedure-related complications. At the 30-day follow-up, there were no serious adverse device events. One death occurred at a 3-month follow-up. The mean LVEF evaluated by MRI improved from 17.7±3.82 % at baseline to 24.94±11.15 % at 6 months post-procedure (p = 0.046). Also, ESV (297.50±67.85 vs 264.79±101.41, p = 0.029), EDV (359.5±71.92 vs 341.56±67.85, p = 0.105), six minutes walking test (339.7±37.6 m vs 370.4±64.8 m, p = 0.184), NYHA functional class (30% Class III and 70% Class IV vs 100% Class II), and KCCQ score (49.7±3.91 vs 79.0±8.1) were improved at 6-month follow-up. Holter monitor data showed no sustained ventricular arrhythmia during the TEAi or follow-up. Conclusions Our results established the safety and feasibility of TEAi using a novel platform, the X-DROP system and paved the way for the clinical application of intramyocardial injection of alginate-hydrogel in patients with HF.Figure 1Figure 2

The effect of cardiosphere-derived cell extracellular vesicles embedded in cardiac matrix hydrogel on cardiac remodeling in a porcine model of ischemic cardiomyopathy

Abstract Background Numerous preclinical studies have confirmed that stem cell-derived extracellular vesicles (EVs) can prevent from negative cardiac remodeling after an ischemic injury, reduce the scar and preserve heart function. It is believed that by increasing the engraftment of the therapeutic product at the target site may bust its efficacy. The use of bioengineering materials such as hydrogels as delivery scaffolds for intramyocardial EVs administration may constitute a minimally invasive therapeutic strategy that improves the efficacy of EVs by increasing their permanence in the tissue. Objective To evaluate the therapeutic efficacy of a new regenerative product constituted by the combination of EVs secreted by cardiosphere-derived cell (CDC-EVs) embedded in a hydrogel-based scaffold made from decellularized cardiac matrix (HDM). Methods Twelve pigs (50% females) with induced ischemic cardiomyopathy were randomly assigned to receive: phosphate-buffer saline (PBS) as control group (n=4), CDC-EVs alone (n=4) and combination of CDC-EVs and HDM (EV-HDM, n=4). Treatment was administered via percutaneous intramyocardial injection in the scar border zone 1-month after myocardial infarction with NOGA system. Animals were followed-up another month and sacrificed. Hearts were excised for histological analysis performed according to the cardiac area: scar (anterior wall), scar border zone and remote zone (inferior wall). Functional evaluation was performed with cardiac magnetic resonance imaging immediately before- and 1-month after treatment. Results Cardiomyocytes size was significantly lower in EV-HDM group compared to control PBS both at scar border (4660± 2088 vs. 5254±2294 pixels; p=0.02) and remote areas (3381±1259 vs. 4237±1256 pixels; p&amp;lt;0.001), while no changes were observed in CDC-EV alone group compared to controls. Interstitial fibrosis was significantly lower in EV-HDM group compared to PBS controls both at scar epicardial side (34% vs. 44%; p=0.01) and remote areas (16% vs. 20%; p&amp;lt;0.01), similarly to CDC-EV vs. PBS (34% at the scar; p&amp;lt;0.01 and 17% at the remote zone; p=0.01). While left ventricular ejection fraction (LVEF) tended to decrease in PBS group from 36% to 32%, it showed an opposite trend in CDC-EV (from 35 % to 38%) and EV-HDM (from 31% to 37%). Conclusion The combined product of EV-HDM reduce cardiac fibrosis and hypertrophy, favorably affecting the remodeling process and may thus increase LVEF in ischemic heart disease. The results of our study fail to clearly demonstrate the superiority of EV-HDM over CDC-EVs alone, although it may exist.

Abstract 12266: CCND2 Modified mRNA Activates the Cell Cycle in Cardiomyocytes and Improves Recovery From Myocardial Infarction in Small and Large Mammals

Background: Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte (CM)-specific overexpression of cyclin D2 (CCND2 in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our Specific Modified mRNA Translation System (SMRTs) can induce CCND2 expression in CMs (CCND2-CMs) and replicate the benefits observed in other studies of CM-specific CCND2 overexpression for myocardial repair. Methods: The CCND2-CM SMRTs consists of two modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the CM-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in CMs, thereby facilitating CM-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the CM-specific expression of luciferase or GFP, in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals. Results: CCND2 was abundantly expressed in cultured, post-mitotic CMs two days after transfection with CCND2-CM SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (e.g., Ki67 and Aurora B kinase). When the GFP-CM SMRTs was intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in CMs but no other cell type. In both the mouse and pig MI models, CM proliferation (on Day 7 and Day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (Days 7 and 28 in mice, Days 10 and 28 in pigs) were significantly higher, and infarcts (Day 28 in both species) were significantly smaller in animals treated with CCND2-CM SMRTs than in any other group that underwent MI induction. Conclusion: Intramyocardial injections of the CCND2-CM SMRTs promoted CM proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with AMI.

Abstract 18612: Stem Cell-Derived Extracellular Matrix as a Treatment for Cardiac Remodeling in Post-MI Hearts: A Pilot Study

Introduction: Post-myocardial infarction (post-MI) cardiac remodeling and decline in cardiac function remains a vexing problem. Tissue engineered extracellular matrix (ECM) has been used in the past but was restricted by the manufactured structure and mechanical limitations. Here, we use a novel ECM substrate generated by self-assembled cells seeded into spherical molds, that are devoid of the hierarchical structure of ECM fabrication, as a treatment for post-MI cardiac remodeling. Hypothesis: Intramyocardial (IM) injection of ECM particles will result in improved cardiac function and reduced infarct size in post-MI hearts. Methods: Human bone marrow mesenchymal stem cells were cultured and ECM particles were isolated, seeded into micro molds, purified, and characterized. ECM particles were suspended at a concentration of 63,400 particles per mL to create an injectable solution. WT mice underwent left anterior descending (LAD) artery ligation followed by IM injection of 10uL of saline control (n=3) or ECM solution (n=7) to the infarcted area (Figure 1A). Mice then underwent post-operative day 3 (POD 3) and POD 7 echocardiogram to determine changes in cardiac function. Results: There was a trend in improving ejection fraction (EF) in ECM injection group compared to control injection in post-MI at POD 3 while no difference was seen at POD 7 (Figure 1B). No difference in fractional shortening (FS) was observed at POD 3 or POD7 (Figure 1C). Echos at POD 14, POD 21, and POD 28 and additional animals will be added to increase study power. Conclusion: IM injection of ECM trends towards mitigating decline in EF at POD3 and does not affect FS in post-MI hearts. These results are likely underpowered (n=3 versus n=7) and may be a portent of significant myocardial protection post-MI. Our ongoing studies (n=10 per group) investigating the mechanisms by which ECM injections assuage ischemic insult to the myocardium may represent a future strategy for clinical therapy.

Abstract 16213: No Cardiac Benefit After Single Dose of Intramyocardial or Intravenous Mesenchymal Stem Cell-Derived Extracellular Vesicles Post-Myocardial Infarction Supports Need for Bioengineered Extracellular Vesicles With Non-Invasive Cardiac Uptake

Introduction: In pre-clinical models, mesenchymal stem cell-derived extracellular vesicles (MSC-EV) have benefits in ischemic CVD. We previously showed that only intramyocardial (IM) injection of MSC-EV, an invasive method of administration, and not intravenous (IV) injection, resulted in reliable cardiac uptake after acute MI. No prior studies have compared the functional outcome of IM versus IV injection after acute MI. Hypothesis: IM injection of MSC-EV will result in improved cardiac outcomes whereas IV injection will not. Aim: To demonstrate the long-term outcomes of IM versus IV MSC-EV after acute MI. Methods: MSC-EV were isolated and characterized. FVB mice underwent left anterior descending coronary artery ligation and subsequent injection of the following: 1) IM saline control (IM-C; n = 8), 2) IM 2*10 9 MSC-EV particles (IM-EV; n = 9), 3) IV saline control (IV-C; n = 8), and 4) IV 2*10 9 MSC-EV particles (IV-EV; n = 8). The mice underwent pre-operative, and post-operative weekly echocardiograms. On post-operative day 28, the mice were euthanized, and the hearts were harvested. Results: Preliminary data showed no significant differences in left ventricular ejection fraction (p = 0.6151) or fractional shortening (p = 0.1135) in the IM-EV and IV-EV groups compared to the controls IM-C and IV-C, respectively, at post-operative day 28 (Figure 1A). There were also no significant differences in infarct size (p = 0.05620) (Figure 1B) or anterior border zone fibrosis (p = 0.6333) (Figure 1C) on Masson-Trichrome staining. Conclusions: Preliminary findings demonstrate that a one-time IM or IV dose of MSC-EV given immediately after acute MI results in no observable effects. Additional MSC-EV doses after MI may produce beneficial effects. Results from our ongoing studies on the creation of stem cells bioengineered to produce MSC-EV with surface cardiac targeting peptides and immune-evasion properties will be presented in August 2023.