Reduction In Scar Size Research Articles

Abstract Mo040: Enhanced neovascularisation does not account for preservation of cardiac function by 11βHSD1 inhibition in a pig model of myocardial infarction

While acute post-myocardial infarction (MI) survival has improved thanks to early intervention, the myocardium still sustains damage that increases the risk of heart failure. Adrenal glucocorticoids (GCs) protect cardiomyocytes immediately after MI, but inhibition of GC regeneration within the heart by 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) during infarct repair prevents subsequent deterioration of ventricular structure and function. In the 11βHSD1-knockout mouse this outcome is associated with enhancement of peri-infarct neovascularisation and prevention of infarct expansion. Here, we tested the hypothesis that pharmacological 11βHSD1 inhibition after MI can preserve function in a translational pig model and investigated the role of neovascularisation. MI was induced in female minipigs via temporary coronary occlusion. A standard clinical therapy (SCT, n =9) group received statin, anti-platelet, βadrenoreceptor antagonist and ACE inhibitor immediatley after MI, and a 11βHSD1i group (n=11) received SCT +11βHSD1 inhibitor (50mg/kg) from 48h after MI until the end of the study. Prior to 11βHSD1i treatment, short-axis cine and late gadolinium-enhanced MRI showed no difference in either infarct mass (4.2±0.4g SCT vs 4.0±0.6g 11βHSD1i) or ejection fraction (EF; 60.6±2.9% SCT vs 56.6±1.8% 11βHSD1i). However, by 28 days after MI, while EF was reduced in SCT pigs to 48±4%, it was maintained in pigs given additional 11βHSD1i treatment (57.9±1.8%, p <0.05 vs SCT). Infarct mass did not differ between the two treatments, suggesting that inhibition of infarct expansion did not account for the improvement in function, this was confirmed by a similar area of infarct collagen in histological analysis. Angiogenesis, represented by an increase in CD31 +ve capillaries, was not enhanced in the infarct or the border zone (BZ) after 11βHSD1i treatment, relative to SCT. While vessel maturation (αSMA +ve vessels) was improved in the infarct compared to the remote myocardium, there was no difference between the two groups. Proteomic pathway analysis of the BZ indicated extracellular matrix organisation as the main pathway regulated by 11βHSD1i compared to SCT. In conclusion, 11βHSD1i after MI successfully preserves cardiac function and structure but the mechanism is independent of scar size reduction or enhanced neovascularisation. Instead, the beneficial effects of 11βHSD1i are likely to result from favourable regulation of collagen processing during scar formation.

Long-Term Protective Effects of Succinate Dehydrogenase Inhibition during Reperfusion with Malonate on Post-Infarction Left Ventricular Scar and Remodeling in Mice.

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 μL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion.

Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats.

Gene therapies to induce cardiomyocyte (CM) cell cycle re-entry have shown a potential to treat subacute ischaemic heart failure (IHF) but have not been tested in the more relevant setting of chronic IHF. Our group recently showed that polycistronic non-integrating lentivirus encoding Cdk1/CyclinB1 and Cdk4/CyclinD1 (TNNT2-4Fpolycistronic-NIL) is effective in inducing CM cell cycle re-entry and ameliorating subacute IHF models and preventing the subsequent IHF-induced congestions in the liver, kidneys, and lungs in rats and pigs. Here, we aim to test the long-term efficacy of TNNT2-4Fpolycistronic-NIL in a rat model of chronic IHF, a setting that differs pathophysiologically from subacute IHF and has greater clinical relevance. Rats were subjected to a 2-h coronary occlusion followed by reperfusion; 4 weeks later, rats were injected intramyocardially with either TNNT2-4Fpolycistronic-NIL or LacZ-NIL. Four months post-viral injection, TNNT2-4Fpolycistronic-NIL-treated rats showed a significant reduction in scar size and a significant improvement in left ventricular (LV) systolic cardiac function but not in the LV dilatation associated with chronic IHF. A mitosis reporter system developed in our lab showed significant induction of CM mitotic activity in TNNT2-4Fpolycistronic-NIL-treated rats. This study demonstrates, for the first time, that TNNT2-4Fpolycistronic-NIL gene therapy induces CM cell cycle re-entry in chronic IHF and improves LV function, and that this salubrious effect is sustained for at least 4 months. Given the high prevalence of chronic IHF, these results have significant clinical implications for developing a novel treatment for this deadly disease.

Macrophage CARD9 mediates cardiac injury following myocardial infarction through regulation of lipocalin 2 expression

Immune cell infiltration in response to myocyte death regulates extracellular matrix remodeling and scar formation after myocardial infarction (MI). Caspase-recruitment domain family member 9 (CARD9) acts as an adapter that mediates the transduction of pro-inflammatory signaling cascades in innate immunity; however, its role in cardiac injury and repair post-MI remains unclear. We found that Card9 was one of the most upregulated Card genes in the ischemic myocardium of mice. CARD9 expression increased considerably 1 day post-MI and declined by day 7 post-MI. Moreover, CARD9 was mainly expressed in F4/80-positive macrophages. Card9 knockout (KO) led to left ventricular function improvement and infarct scar size reduction in mice 28 days post-MI. Additionally, Card9 KO suppressed cardiomyocyte apoptosis in the border region and attenuated matrix metalloproteinase (MMP) expression. RNA sequencing revealed that Card9 KO significantly suppressed lipocalin 2 (Lcn2) expression post-MI. Both LCN2 and the receptor solute carrier family 22 member 17 (SL22A17) were detected in macrophages. Subsequently, we demonstrated that Card9 overexpression increased LCN2 expression, while Card9 KO inhibited necrotic cell-induced LCN2 upregulation in macrophages, likely through NF-κB. Lcn2 KO showed beneficial effects post-MI, and recombinant LCN2 diminished the protective effects of Card9 KO in vivo. Lcn2 KO reduced MMP9 post-MI, and Lcn2 overexpression increased Mmp9 expression in macrophages. Slc22a17 knockdown in macrophages reduced MMP9 release with recombinant LCN2 treatment. In conclusion, our results demonstrate that macrophage CARD9 mediates the deterioration of cardiac function and adverse remodeling post-MI via LCN2.

Higher Improvement of Cardiac Function Following Myocardial Infarction using Menstrual Blood Stromal/Stem Cells (MenSCs) Suspended in Conditioned Medium versus Conditioned Medium Alone in Rat Model.

To evaluate the efficiency of Menstrual blood Stromal/Stem Cells (MenSCs) administration in Myocardial Infarction (MI), the effects of MenSCs and their derived conditioned Medium (CM) on cardiac function in MI rat model was assessed. Animals were divided into four groups including sham group, MI group, MenSCs derived CM group (CM group), and MenSCs suspended in CM (MenSCs+CM) group. The injection of different groups was carried out 30 min after ligation of left anterior descending coronary artery into the infarct border zone. The results showed a significant reduction in scar size after injection of MenSCs+CM compared to MI group. Ejection fraction and fractional shortening of MenSCs+CM group were higher than CM and MI group at day 28. Administration of MenSCs+CM led to much more survival of cardiomyocytes, and prevention of meta-plastic development. Moreover, human mitochondrial transfer from MenSCs to cardiomyocytes was seen in group treated by MenSCs+CM. Indeed, MenSCs+CM treatment evoked nuclear factor-κB (NF-κB) down-regulation more than other treatments. MenSCs+CM treatment could significantly ameliorate cardiac function by different mechanisms including inhibition of cartilaginous metaplasia, inhibition of NF-κB and mitochondrial transfer.

The effect of percutaneous tract dilation technique on renal parenchymal trauma: An experimental in vivo study on a porcine model.

The purpose of this study was to evaluate renal parenchymal trauma of two-step dilation compared to the conventional Amplatz gradual dilation during percutaneous nephrolithotomy on a porcine model. A nonpapillary percutaneous access tract was established under fluoroscopic guidance in both kidneys of four female pigs. On the right kidney of each pig, gradual dilation was performed using an Amplatz dilator set with a gradual dilation to 30 Fr, whereas on the left, a two-step dilation was utilized using only 16 Fr and 30 Fr dilators. Two of the animals were euthanized immediately after the procedure and the remaining two 1 month later. The pigs that were kept alive underwent a contrast-enhanced computed tomography immediately, 15, and 30 days postoperatively. A dimercaptosuccinic acid (DMSA) scintigraphy and single-photon emission computed tomography-computed tomography (CT) were also performed after the last CT and afterward, the pigs were sacrificed. All kidneys were harvested for pathohistological examination. The follow-up radiologic imaging showed similar parenchymal damage caused by the compared dilation techniques and an expected reduction in scar size in the later scans. No scar was identified by DMSA in any kidney. Gross and microscopic examinations conducted both on the kidneys that were harvested immediately after the procedure and the ones from the animals that were left to heal, revealed no significant differences in tissue damage, grade of fibrosis, or inflammation depending on the dilation method. Our study showed no inferior outcomes caused by two-step dilation compared to gradual dilation regarding renal parenchymal damage following a nonpapillary puncture. In fact, postoperative imaging findings suggested a trend toward better healing and less scar tissue when the two-step method was used.

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1β release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow–derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

Abstract 10492: Preclinical Evaluation of Transient and Cardiomyocyte Specific Gene Therapy for the Treatment of Subacute Ischemic Heart Failure

Rationale: Treatment of heart failure is limited due to the limited regenerative capacity of cardiomyocytes. Our group has shown that ectopic expression of Cdk1/CyclinB1 and Cdk4/CyclinD1 (named 4F) via adenovirus vector promotes cardiomyocyte proliferation in 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction (MI). Objective: To introduce 4F specifically and transiently into cardiomyocytes to induce selective cardiomyocyte regeneration and limit any tumorigenic potential in other organs. Methods and Results: A polycistronic non-integrating lentivirus encoding 4F, each driven by a TNNT2 promoter (TNNT2-4F-NIL), was generated to induce transient and specific expression of 4F cardiomyocytes. TNNT2-4F-NIL promotes proliferation of 15% of cardiomyocytes in vivo after MI as assessed in lineage-tracing mice (α-MHC-Cre-MADM). Rats and pigs were subjected to coronary occlusion/reperfusion to induce MI. One week later, animals were injected (intramyocardially) with either TNNT2-4F-NIL or control-NIL. Four weeks after injection, TNNT2-4F-NIL treated rats and pigs showed a significant improvement in left ventricular ejection fraction (EF) compared to controls (TNNT2-4F-NIL rats 54.3±1.78% vs control-NIL 42.97±2.60%; n=9/group, p =0.003, TNNT2-4F-NIL pigs 37±2.3% vs control-NIL 28.4±1.2%; n=6/group, p =0.01). Histological analyses showed a 25-30% reduction in scar size in both rats and pigs treated with TNNT2-4F-NIL vs. control-NIL (p&lt;0.05). To assess the long-term efficacy and safety of TNNT2-4F-NIL, a 4-months follow-up experiment after MI and virus treatment was performed during which cardiac function was assessed monthly. TNNT2-4F-NIL treated rats showed sustained improvement in cardiac function (EF in TNNT2-4F-NIL 48.4±3% vs in control-NIL 33.6±3%; n=6/group, p =0.008). There was no obvious development of arrhythmias and no evidence of oncogenicity or toxicity in other organs in TNNT2-4F-NIL treated rats or in the control group. Conclusion: This study provides a novel approach to treat heart failure, with the use of transient and cardiomyocyte-specific gene therapy that promotes long-term functional improvement after a single treatment with no obvious adverse effect.

Cortical bone stem cell-derived exosomes' therapeutic effect on myocardial ischemia-reperfusion and cardiac remodeling.

Heart failure is the one of the leading causes of death in the United States. Heart failure is a complex syndrome caused by numerous diseases, including severe myocardial infarction (MI). MI occurs after an occlusion of a cardiac artery causing downstream ischemia. MI is followed by cardiac remodeling involving extensive remodeling and fibrosis, which, if the original insult is severe or prolonged, can ultimately progress into heart failure. There is no "cure" for heart failure because therapies to regenerate dead tissue are not yet available. Previous studies have shown that in both post-MI and post-ischemia-reperfusion (I/R) models of heart failure, administration of cortical bone stem cell (CBSC) treatment leads to a reduction in scar size and improved cardiac function. Our first study investigated the ability of mouse CBSC-derived exosomes (mCBSC-dEXO) to recapitulate mouse CBSCs (mCBSC) therapeutic effects in a 24-h post-I/R model. This study showed that injection of mCBSCs and mCBSC-dEXOs into the ischemic region of an infarct had a protective effect against I/R injury. mCBSC-dEXOs recapitulated the effects of CBSC treatment post-I/R, indicating exosomes are partly responsible for CBSC's beneficial effects. To examine if exosomes decrease fibrotic activation, adult rat ventricular fibroblasts (ARVFs) and adult human cardiac fibroblasts (NHCFs) were treated with transforming growth factor β (TGFβ) to activate fibrotic signaling before treatment with mCBSC- and human CBSC (hCBSC)-dEXOs. hCBSC-dEXOs caused a 100-fold decrease in human fibroblast activation. To further understand the signaling mechanisms regulating the protective decrease in fibrosis, we performed RNA sequencing on the NHCFs after hCBSC-dEXO treatment. The group treated with both TGFβ and exosomes showed a decrease in small nucleolar RNA (snoRNA), known to be involved with ribosome stability.NEW & NOTEWORTHY Our work is noteworthy due to the identification of factors within stem cell-derived exosomes (dEXOs) that alter fibroblast activation through the hereto-unknown mechanism of decreasing small nucleolar RNA (snoRNA) signaling within cardiac fibroblasts. The study also shows that the injection of stem cells or a stem-cell-derived exosome therapy at the onset of reperfusion elicits cardioprotection, emphasizing the importance of early treatment in the post-ischemia-reperfusion (I/R) wounded heart.

In vivo-wound healing studies of sodium thiosulfate gel in rats

Sodium Thiosulfate (STS) is already reported as an antioxidant, anti-inflammatory agent with antiseptic, antifungal properties. The search for an ideal antiseptic still continues, which is lethal to all types of bacteria and their spores and sustain the activity for a longer time without any harm to the host tissue. The aim of the present study is to evaluate the effect of STS on curing of wounds in rats when compared to Betadine. We developed topical gels having 6% and 12% STS. The effects of STS on wound healing rate of Rats were evaluated against Betadine as positive control. Wounds of control group, selected as Group 1 was treated with normal saline (0.2 ml), twice a day. Reference standard control, designated as Group 2 rats were given with 0.2 ml Betadine twice a day. Rats in Groups 3 and 4 were treated with 0.2 ml of STS gel (6% or 12% respectively) twice a day. In our study, STS formulation has proved to be a safe and efficient wound healing product. It has a neutral pH and longer half life (>12 months). Higher STS dose of 12% proved to have a wound curing rate equivalent to that of Betadine. On 11th Day, 97 ± 0.79% healing was achieved with Betadine and 98 ± 0.67% with 12% STS Gel (∗P < 0.05). Microscopic images of H&E stained skin tissue from animals treated with Betadine and 12% STS formulation showed a reduction in scar size, lesser amount of inflammatory cells, higher fibroblasts and blood vessels, with considerable collagen accumulation. Furthermore, a significant enhancement in the levels of GPx, CAT and SOD was observed in the tissue at the wound site of the treated group. The IL 10 levels in both groups of STS-treated rats was increased, whereas, TNF-α levels were reduced significantly in tissue homogenate compared with control. Thus, this study shows the wound-healing performance of STS formulation. Further studies are necessary to understand the real mechanism of how STS formulation heals wounds.

Abstract 223: Yap and Its Homolog Wwtr1 Are Regulators of Myofibroblast Activation Following Ischemic Injury

Introduction: Following ischemic injury in adult mammals, cardiac fibroblasts differentiate into myofibroblasts and promote secretion of matrix fibers. Myofibroblast activation is critical for initial scar formation and preventing heart rupture, however, extended activity can lead to heart failure progression. Thus, there is a need to identify the mechanisms mediating persistent activation of myofibroblasts to prevent excessive fibrosis and adverse cardiac remodeling. Here we demonstrate that Hippo-Yap pathway offers a target for modulating myofibroblast activation and thus the fibrotic response. Methods and Results: We tested the hypothesis that Yap and its homolog Wwtr1 (known as ‘Taz’) are regulators of myofibroblast activation following ischemic injury. We implemented a Cre-lox system whereby Yap alone or both Yap and Wwrt1 were depleted using an inducible Cre expressed under a myofibroblast specific promoter ( Postn MCM ). Following permeant ligation of the left anterior descending artery in adult mice, we found that myofibroblast depletion of Yap alone resulted in a significant reduction in left ventricular dilation 28 days post injury (dpi) and decreased proliferation of scar associated cells. Strikingly, myofibroblast specific depletion of Yap and one copy of Wwrt1 resulted in further attenuation of left ventricular dilation as well as improved fractional shortening and ejection fraction at 28 and 60 dpi. Histological assessment revealed that depletion of both Yap and Wwrt1 resulted in greater than 50% reduction in scar size (by midline) at 60 dpi. RNAseq of whole hearts collected at 4 dpi suggested that Hippo-Yap pathway expression specifically in myofibroblasts facilitates immune cell recruitment in the heart. Collectively These data illustrate a role for Hippo-Yap signaling mediating myofibroblast activity and immune cell coordination following injury and therefore cardiac fibrosis and remodeling. Conclusions: Our data demonstrate that endogenous Yap and Wwrt1 deletion in myofibroblasts suppresses the fibrotic response, mediates inflammation, and improves cardiac function after ischemic injury. These results therefore offer a regulatory pathway that can be targeted therapeutically to prevent progressive heart failure.

Cardiac Revascularization Post Myocardial Infarction Enhances Remuscularization and Improves Function

BackgroundHuman induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) offer an unprecedented opportunity to remuscularize infarcted human hearts. However, studies show that the majority of hiPSC‐CMs die post transplantation into the ischemic environment, limiting their regenerative potential and clinical application. Death of transplanted CMs occurs in the first few days post‐transplantation due to ischemia. Thus, attempts have been made to promote blood perfusion (i.e. addition of endothelial cells and/or angiogenic factors). However, these approaches require weeks for new vessels to form and to carry blood compared to the rapid death of transplanted CMs (2–3 days).PurposeOur goal was to improve the vascularization of the ischemic hearts to improve the survival of hiPSC‐CMs and increase heart remuscularization and function.MethodsWe performed left anterior descending artery (LAD) ligation in immunocompromised rats to model myocardial infarction. To improve vascularization, we used an innovative strategy consisting of ready‐made microvessels isolated from adipose tissue that form a vasculature and carry blood within the first days post subcutaneous implantation. We have co‐implanted ready‐made microvessels with hiPSC‐CMs by intra‐myocardial injection 2 weeks post LAD ligation. Cardiac function was assessed at 0, 2 and 4 weeks post‐implantation by echocardiography and by pressure‐volume loop at the endpoint (4 weeks). Immunohistochemistry and blood perfusion studies were performed at 1 and 4 weeks post‐implantation.ResultsCompared to hiPSC‐CM transplantation alone, microvessels promoted a ~600% increase in hiPSC‐CM survival with significant reduction in scar size. Echocardiography and pressure–volume (PV) loop analysis performed 4 weeks post‐MI revealed that hiPSC‐CM transplantation attenuated post‐infarct ventricular dilation and enhanced left ventricular contractility by demonstrating a significant improvement in fractional shortening (FS), ejection fraction (EF) as well as other functional parameters (end‐systolic volume, end‐diastolic volume, dP/dt max and min, Tau). Remarkably, co‐transplantation of hiPSC‐CMs with microvessels showed significantly superior functional recovery compared to hiPSC‐CMs alone in all the parameters assessed. Microvessels showed remarkable persistence and integration at both early and late time points, resulting in significantly faster blood perfusion and higher vessel density in the grafts.ConclusionThese findings provide a novel approach to cell‐based therapies for myocardial infarction whereby incorporation of ready‐made microvessels can serve as a personalized delivery system to improve functional outcomes in cell replacement therapies post‐myocardial infarction.Support or Funding InformationCanadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health grant #137352 and #PJT153160. Discovery grant from the Natural Sciences and Engineering Research Council (NESERC): RGPIN 06621‐2017.

Multi-phase catheter-injectable hydrogel enables dual-stage protein-engineered cytokine release to mitigate adverse left ventricular remodeling following myocardial infarction in a small animal model and a large animal model

Although ischemic heart disease is the leading cause of death worldwide, mainstay treatments ultimately fail because they do not adequately address disease pathophysiology. Restoring the microvascular perfusion deficit remains a significant unmet need and may be addressed via delivery of pro-angiogenic cytokines. The therapeutic effect of cytokines can be enhanced by encapsulation within hydrogels, but current hydrogels do not offer sufficient clinical translatability due to unfavorable viscoelastic mechanical behavior which directly impacts the ability for minimally-invasive catheter delivery. In this report, we examine the therapeutic implications of dual-stage cytokine release from a novel, highly shear-thinning biocompatible catheter-deliverable hydrogel. We chose to encapsulate two protein-engineered cytokines, namely dimeric fragment of hepatocyte growth factor (HGFdf) and engineered stromal cell-derived factor 1α (ESA), which target distinct disease pathways. The controlled release of HGFdf and ESA from separate phases of the hyaluronic acid-based hydrogel allows extended and pronounced beneficial effects due to the precise timing of release. We evaluated the therapeutic efficacy of this treatment strategy in a small animal model of myocardial ischemia and observed a significant benefit in biological and functional parameters. Given the encouraging results from the small animal experiment, we translated this treatment to a large animal preclinical model and observed a reduction in scar size, indicating this strategy could serve as a potential adjunct therapy for the millions of people suffering from ischemic heart disease.

4938Left ventricular global longitudinal strain recovery predicts scar size reduction and systolic remodelling post ST-elevation myocardial infarction

Abstract Background Left ventricular (LV) strain has prognostic utility following ST-elevation myocardial infarction (STEMI); however, serial changes in LV strain has not been evaluated post-infarct. We sought to determine the relationship between post-STEMI transthoracic echocardiographic (TTE) LV global longitudinal strain (GLS) and cardiac magnetic resonance (CMR) imaging derived scar size and LV systolic remodelling. Methods Following revascularisation, 172 first STEMI patients (85% male, 56.9±10.7 years) had paired TTE for GLS, and CMR to evaluate scar size and LV systolic function at baseline (2–7 days) and follow-up (8–10 weeks). Patients were divided into 3 groups according to absolute baseline GLS: group 1 (GLS ≥16%), group 2 (12%&lt; GLS &lt;16%), group 3 (GLS ≤12%). GLS recovery was defined as ≥10% increase in GLS at follow-up, excluding patients with normal baseline GLS. LV systolic adverse remodelling was defined as ≥15% increase in LVESV. LV systolic reverse remodelling was defined as ≥15% decrease in LVESV. Scar reduction was defined as ≥30% decrease in scar size. Results Group 1 and 2 had smaller follow-up scar size and higher LVEF compared to group 3 (p&lt;0.0001 for both, see table). There was no difference in scar size reduction or systolic reverse remodeling among the baseline GLS groups (p&gt;0.05 for both). Importantly, no patients from group 1 demonstrated systolic adverse remodelling. Relative change in GLS is significantly correlated with changes in LVEF (r=0.354, p&lt;0.0001) and scar size (r=−0.262, p&lt;0.0001), see figure. On multivariate binary logistic analysis, patients who demonstrated GLS recovery had greater reduction in scar size (OR=2.77 (1.09–7.01), p=0.032) and LV systolic reverse remodelling (OR=9.63 (1.21–76.41), p=0.032). Follow-up parameters within GLS groups All patients (n=172) Group 1 (n=47) Group 2 (n=72) Group 3 (n=53) Follow-up GLS, % 16.02±3.44 19.38±1.90 16.36±2.09 12.57±2.69 GLS recovery, n 110 (64%) 19 (40%) 53 (74%) 38 (72%) Follow-up scar size, % 7.67±5.40 5.01±3.38 6.27±3.73 12.02±6.24 Follow-up LVEF, % 51.80±10.20 57.83±6.95 54.14±8.02 43.26±9.83 Data presented as mean ± SD or n (%). Correlation graphs for change in GLS Conclusion Stratification of STEMI patients by baseline GLS was a determinant of CMR scar size as well as LV systolic function. However, the evaluation of GLS recovery could provide additional insights into reduction in scar size and LV systolic remodelling, both important prognostic markers. Thus, echocardiographic serial GLS evaluation may be a relevant non-invasive parameter, that is cheaper and more widely available for monitoring STEMI patients and guiding therapy.

Abstract 418: Ready-made Microvessels Robustly Integrate Into the Infarcted Coronary Vasculature Promoting Graft Perfusion, Enhancing Cardiac Remuscularization and Function

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an unprecedented opportunity to remuscularize infarcted hearts. However, the majority of hiPSC-CMs die post transplantation into the ischemic environment, limiting their regenerative potential. CM death occurs in the first few days post-transplantation due to ischemia. Attempts to promote intramyocardial vascularization (i.e. delivery of growth factors or cells) have not led to significant improvements due to poor cell retention and the long time required for new vessels to form and carry blood compared to the rapid death of transplanted CMs. Here, we used ready-made microvessels harvested from adipose tissue - that form a vasculature and carry blood within the first days post subcutaneous implantation - to re-vascularize ischemic rat hearts and improve hiPSC-CM survival. We performed left anterior descending artery ligation in immunocompromised rats to model MI. hiPSC-CMs (10 x10 6 ) with (CM+V) or without (CM-only, control) microvessels were delivered by intra-myocardial injection 2 weeks post MI. Cardiac function was assessed by echocardiography and pressure-volume loop. Delivery of microvessels from GFP rats allowed assessment of microvessel persistence. Compared to hiPSC-CM transplantation alone, microvessels promoted a 6-fold increase in hiPSC-CM survival, with reduction in scar size and a significantly superior functional recovery. While delivery of CM-only stabilized the heart preventing further fractional shortening decline, delivery of CM+V resulted in reversal of fractional shortening loss. This was supported by PV-loop data (ejection fraction: sham, ~20%; CM-only, ~31%; CM+V, ~39%; 4 wks). Moreover, microvessels showed unprecedented persistence and integration (&gt;60%, 4 wks), resulting in 2-fold increase in vessel area and graft perfusion (as early as day 5 post-transplantation). This was achieved despite the very low number of cells (~2 x10 5 ) delivered in the form of microvessels. These findings provide a novel approach to cell-based therapies for MI whereby incorporation of ready-made microvessels can serve as a personalized delivery system to improve functional outcomes in cell replacement therapies.

Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair.

Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.

Insulin‐like growth factor 1 preserves cardiac function after myocardial infarction by affecting myeloid cells

Insulin‐like growth factor (IGF1) controls growth and metabolism of many cell types. In addition, IGF1 has been shown to provide cardioprotective effects after acute myocardial infarction (AMI). However, the cell type and mechanisms involved are not yet fully understood.Mice were exposed to 45 min LAD occlusion, followed by 4 weeks reperfusion. At the end of ischemia mice were injected with vehicle (Con) or IGF1 (40 ng/g ip), followed by continues treatment for 3 days via osmotic mini pumps (1 μg/g/d sc.). Cardiac function, determined by echocardiography at baseline and 1 and 4 weeks after ischemia, showed no differences at baseline in all mouse lines studied. IGF1 treatment improved cardiac function after 1 and 4 weeks in wild‐type mice ((EF, week 4: 49±4% (IGF1) vs 36±8% (Con)), but also in inducible cardiomyocyte‐specific IGF1‐receptor (IGF1R) KO mice (48±5% (IGF1) vs 35±5% (Con)). However, in myeloid cell specific IGF1R KO mice IGF1 was no longer protective (36±8% (IGF1) vs 38±8% (Con)), indicating that myeloid cells are responsible for the protective effect of IGF1 after MI. In addition, micro‐array analysis of the infarct area of the heart showed that mostly myeloid cell related pathways were altered in IGF treated mice 1 and 2 days after AMI.To look further into the mechanism involved, in vitro and subacute experiments were performed. FACS and qPCR analysis showed that treatment of monocyte derived macrophages with IGF1 induced macrophage polarization to macrophages with a reparative M2‐like phenotype, characterized by an increase in mannose receptor (CD206), arginase and resistin‐like α. Also, in vivo FACS analysis of the heart showed an increase in M2‐like (CD206+) macrophages in IGF1 treated mice 3 days after AMI (152 777±13510 (IGF1) vs 108 071±14023 (Con) cells per heart) without affecting total macrophage number. In these mice, no effects on the amount of lymphocytes or monocytes were observed in blood or heart. A trend towards an increased number of neutrophils in the heart was seen after IGF treatment (272 120±42478 (IGF1) vs 198 122±31741 (Con) cells per heart), without an effect on neutrophil amount in blood. In addition, one week after AMI, i.e. a timepoint where functional cardiac improvement was observed in echo analysis, histological analysis of scar size showed a significant reduction in scar size after IGF1 treatment (9.2±4% (IGF1) vs. 14.7±3.9% (Con) of left ventricle). This was accompanied by an increased vessel formation in the scar (654±132 (IGF1) vs 495±94 (Con) CD31+ cells/mm2) and borderzone (1676±72 (IGF1) vs 1467±110 (Con)) after IGF1 treatment.Interestingly, short term no protective effects of IGF1 were observed. IGF1 treatment did not reduce infarct size 2 hours after AMI (38±10% (IGF1) vs. 39±11% (Con) of left ventricle).Short term IGF1 treatment improves cardiac function in the subacute phase after AMI by affecting myeloid cells. This effect was independent of the IGF1R in cardiomyocytes. IGF1 treatment increases the amount of reparative M2 macrophages, and may thereby improve vasculature and reduce scar size.Support or Funding InformationFunded by SFB1116/A06This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non-inferiority of microencapsulated mesenchymal stem cells to free cells in cardiac repair after myocardial infarction: A rationale for using paracrine factor(s) instead of cells.

A major translational barrier to the use of stem cell (SC)-based therapy in patients with myocardial infarction (MI) is the lack of a clear understanding of the mechanism(s) underlying the cardioprotective effect of SCs. Numerous paracrine factors from SCs may account for reduction in infarct size, but myocardial salvage associated with transdifferentiation of SCs into vascular cells as well as cardiomyocyte-like cells may be involved too. In this study, bone marrow-derived rat mesenchymal SC (MSCs) were microencapsulated in alginate preventing viable cell release while supporting their secretory phenotype. The hypothesis on the key role of paracrine factors from MSCs in their cardioprotective activity was tested by comparison of the effect of encapsulated vs free MSCs in the rat model of MI. Intramyocardial administration of both free and encapsulated MSCs after MI caused reduction in scar size (12.1±6.83 and 14.7±4.26%, respectively, vs 21.7±6.88% in controls, P=0.015 and P=0.03 respectively). Scar size was not different in animals treated with free and encapsulated MSC (P=0.637). These data provide evidence that MSC-derived growth factors and cytokines are crucial for cardioprotection elicited by MSC. Administration of either free or encapsulated MSCs was not arrhythmogenic in non-infarcted rats. The consistency of our data with the results of other studies on the major role of MSC secretome components in cardiac protection further support the theory that the use of live, though encapsulated, cells for MI therapy may be replaced with heart-targeted-sustained delivery of growth factors/cytokines.

Mechanical and Chemical Predifferentiation of Mesenchymal Stem Cells Into Cardiomyocytes and Their Effectiveness on Acute Myocardial Infarction.

Myocardial infarction is one of the leading causes of death all over the world. Mesenchymal stem cells (MSCs) transplantation has shown a promising potential to recovery of ischemic heart disease due to their capability in differentiating into cardiac cells. However, various investigations have been performed to optimize the efficacy of cardiac cell therapy in recent years. Here, we sought to interrogate the effect of autologous transplantation of undifferentiated and predifferentiated adipose and bone marrow-derived MSCs in a rabbit model of myocardial infarction and also to investigate whether cardiac function could be improved by mechanically induced MSCs via equiaxial cyclic strain. The two sources of MSCs were induced toward cardiomyocyte phenotype using mechanical loading and chemical factors and thereafter injected into the infarcted myocardium of 35 rabbits. Echocardiography and histopathology studies were used to evaluate cardiac function after 2 months. The results demonstrated significant scar size reduction and greater recovery of left ventricle ejection fraction after transplantation of predifferentiated cells, though the differences were not significant when comparing mechanically with chemically predifferentiated MSCs. Thus, although there was no significant improvement in infarcted myocardium between chemically and mechanically predifferentiated MSCs, mechanically induced cells are more preferred due to lack of any chemical intervention and cost reasonableness in their preparation method. Outcomes of this study may be useful for developing future therapeutic strategies, however long-term assessments are still required to further examine their effectiveness.