Role of lymphatic network in cardiac repair post myocardial infarction

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-β1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-β/Smad2/3 signaling pathway.

Background: ECM proteins are instrumental for angiogenesis, which plays momentous roles during development and repair in various organs, including post cardiac insult. After a screening based on an open access RNA-seq database, we identified Nephronectin (NPNT), an extracellular protein, might be involved in cardiac repair post myocardial infarction (MI). However, the specific impact of nephronectin during cardiac repair in MI remains elusive.Methods and Results: In the present study, we established a system overexpressing NPNT locally in mouse heart by utilizing a recombinant adeno-associated virus. One-to-four weeks post MI induction, we observed improved cardiac function, limited infarct size, alleviated cardiac fibrosis, with promoted angiogenesis in infarct border zone in NPNT overexpressed mice. And NPNT treatment enhanced human umbilical vascular endothelial cell (HUVEC) migration and tube formation, putatively through advocating phosphorylation of EGFR/JAK2/STAT3. The migration and capillary-like tube formation events could be readily revoked by EGFR or STAT3 inhibition. Notably, phosphorylation of EGFR, JAK2 and STAT3 were markedly upregulated in AAV2/9-cTnT-NPNT-treated mice with MI.Conclusions: Our study thus identifies the beneficial effects of NPNT on angiogenesis and cardiac repair post MI by enhancing the EGFR/JAK2/STAT3 signaling pathway, implying the potential therapeutic application of NPNT on myocardial dysfunction post MI.

Myocardial healing following myocardial infarction (MI) toward either functional tissue repair or excessive scarring/heart failure, may depend on a complex interplay between nervous and immune system responses, myocardial ischemia/reperfusion injury factors, as well as genetic and epidemiological factors. Hence, enhancing cardiac repair post MI may require a more patient-specific approach targeting this complex interplay and not just the heart, bearing in mind that the dysregulation or modulation of just one of these systems or some of their mechanisms may determine the outcome either toward functional repair or toward heart failure.In this review we have elected to focus on existing preclinical and clinical in-vivo studies aimed at testing novel therapeutic approaches targeting the nervous and immune systems to trigger myocardial healing toward functional tissue repair. To this end, we have only selected clinical and preclinical in-vivo studies reporting on novel treatments targeting neuro-immune systems to ultimately treat MI. Next, we have grouped and reported treatments under each neuro-immune system. Finally, for each treatment we have assessed and reported the results of each clinical/preclinical study and then discussed their results collectively. This structured approach has been followed for each treatment discussed. To keep this review focused, we have deliberately omitted to cover other important and related research areas such as myocardial ischemia/reperfusion injury, cell and gene therapies as well as any ex-vivo and in-vitro studies.The review indicates that some of the treatments targeting the neuro-immune/inflammatory systems appear to induce beneficial effects remotely on the healing heart post MI, warranting further validation. These remote effects on the heart also indicates the presence of an overarching synergic response occurring across the nervous and immune systems in response to acute MI, which appear to influence cardiac tissue repair in different ways depending on age and timing of treatment delivery following MI. The cumulative evidence arising from this review allows also to make informed considerations on safe as opposed to detrimental treatments, and within the safe treatments to ascertain those associated with conflicting or supporting preclinical data, and those warranting further validation.

To determine the effect of the pre-treatment of mesenchymal stem cells (MSCs) with minocycline on the expression of antioxidant genes and cardiac repair post myocardial infarction (MI) in rats. Rat bone marrow derived MSCs were used in the study. Cytotoxicity of minocycline in MSCs was determined using JC1 assay to identify a safe drug dose for further experiments. The MSCs were pre-treated with 1.0 µM minocycline for 24 hours and then treated with hydrogen peroxide (H2O2), after that mRNA was isolated and the expression levels of antioxidant genes including peroxiredoxin, glutathione peroxidase, and superoxide dismutase were determined. Finally, minocycline pre-treated MSCs were used to treat rats induced with MI by the ligation of left anterior descending coronary artery. The cardiac function was evaluated at two and four weeks post MI using echocardiography. At 1.0 µM concentration, minocycline was found to be safe for MSCs and used for subsequent experiments. Minocycline pre-treatment was found to up regulate several antioxidant genes in oxidatively stressed MSCs. Furthermore, minocycline pre-treated MSCs displayed greater improvement in cardiac left ventricular function at two and four-weeks post MI as compared to untreated rats. Pre-treatment of MSCs with minocycline enhances the expression of antioxidant genes and promotes their capability to repair cardiac function after MI.

A comprehensive understanding of the genome-wide regulatory landscape of the cardiac tissues post-myocardial infarction (MI) is still lacking. We therefore integrated single-cell RNA sequencing (scRNA-seq) and single-cell for transposase-accessible chromatin sequencing (scATAC-seq) to elucidate the epigenetic landscape of the heart post-MI. We established MI mice through ligation of the left anterior descending coronary artery, and obtained cardiac tissues from mice at 1-, 3-, 7-, and 14-days post-MI. Integrative analyses of the scRNA-seq and scATAC-seq data revealed the presence of two novel fibroblast subpopulations in the cardiac tissues of MI mice, termed GATA-binding protein 5/ISL LIM Homeobox 1 (GATA5/ISL1)+ fibroblasts and GLI family zinc finger 3 (Gli3)high fibroblasts. The GATA5/ISL1+ fibroblasts were characterized by fibroblast and cardiomyocyte signatures and were found to play a crucial role in cardiac repair post-MI. Moreover, adenoviral-mediated overexpression of GATA5 and ISL1 ameliorated cardiac function and attenuated myocardial fibrosis in the MI mice. RNA sequencing confirmed that GATA5 and ISL1 co-regulate Wnt signalling pathway to promote the transformation of fibroblasts into functional cardiomyocytes. Furthermore, analysis of the human cardiac tissues of MI patients also revealed the presence of GATA5/ISL1+ fibroblasts in the scar tissues, suggesting their crucial role in cardiac tissue repair post-MI. In addition, proteomic analyses revealed enhanced cardiac repair and development signalling in the GATA5/ISL1-overexpressing human cardiac fibroblasts. The study provides novel perspectives on the mechanisms of myocardial injury and repair at the single-cell level and indicates the potential role of GATA5 and ISL1 as therapeutic targets for MI treatment.

SummaryTissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment.

Epicardial placement of mesenchymal stromal cells (MSCs) is a promising strategy for cardiac repair post-myocardial infarction, but requires the design of biomaterials to maximise the retention of donor cells on the heart surface and control their phenotype. To this end, we propose the use of a poly(2-alkyl-2-oxazoline) (POx) derivative, based on 2-ethyl-2-oxazoline and 2-butenyl-2-oxazoline. This POx polymer can be cured rapidly (less than 2 min) via photo-irradiation due to the use of di-cysteine cell degradable peptides. We report that the cell-degradable properties of the resulting POx hydrogels enables the regulation of cell protrusion in corresponding 3D matrices and that this, in turn, regulates the secretory phenotype of MSCs. In particular, the expression of pro-angiogenic genes was upregulated in partially cell-degradable POx hydrogels. Improved angiogenesis was confirmed in an in vitro microfluidic assay. Finally, we confirmed that, owing to the excellent tissue adhesive properties of thiol-ene crosslinked hydrogels, the epicardial placement of MSC-loaded POx hydrogels promoted the recovery of cardiac function and structure with reduced interstitial fibrosis and improved neovascular formation in a rat myocardial infarction model. This report demonstrates that engineered synthetic hydrogels displaying controlled mechanical, cell degradable and bioactive properties are particularly attractive candidates for the epicardial placement of stem cells to promote cardiac repair post myocardial infarction.

BackgroundMyocardial microvascular loss after myocardial infarction (MI) remains a therapeutic challenge. Autologous stem cell therapy was considered as an alternative; however, it has shown modest benefits due to the impairing effects of cardiovascular risk factors on stem cells. Allogenic adipose-derived stem cells (ASCs) may overcome such limitations, and because of their low immunogenicity and paracrine potential may be good candidates for cell therapy. In the present study we investigated the effects of allogenic ASCs and their released products on cardiac rarefaction post MI.MethodsPig subcutaneous adipose tissue ASCs were isolated, expanded and GFP-labeled. ASC angiogenic function was assessed by the in-vivo chick chorioallantoic membrane (CAM) model. Pigs underwent MI induction and 7 days after were randomized to receive: allogenic ASCs (intracoronary infusion); conditioned media (CM; intravenous infusion); ASCs + CM; or PBS/placebo (control). Cardiac damage and function were monitored by 3-T cardiac magnetic resonance imaging upon infusion (baseline CMR) and 1 and 3 weeks thereafter. We assessed in the myocardium: microvessel density; angiogenic markers (CD105, CD31, TF, VEGFR2, VEGFR1, vWF, eNOS, CD62); collagen deposition; and reparative fibrosis (TGFβ/TβRII/collagen). Differential proteomics of ASCs and CM was performed to characterize the ASC protein signature.ResultsCAM indicated a significant ASC proangiogenic capacity. In pigs after MI, only PBS/placebo animals displayed an impaired cardiac function 3 weeks after infusion (p < 0.05 vs baseline). Administration of ASCs + CM significantly enhanced neovessel formation and favored cardiac repair post MI (p < 0.05 vs the other groups). Molecular markers of angiogenesis were significantly upregulated both at transcriptional and protein levels (p < 0.05). The in-silico bioinformatics analysis of the ASC and CM proteome (interactome) indicated activation of a coordinated protein network involved in the formation of microvessels and the resolution of rarefaction.ConclusionCoadministration of allogenic ASCs and their CM synergistically contribute to the neovascularization of the infarcted myocardium through a coordinated upregulation of the proangiogenic protein interactome.

Myeloid-derived growth factor (Mydgf), a paracrine protein secreted by bone marrow-derived monocytes and macrophages, was found to protect against cardiac injury following myocardial infarction (MI) in adult mice. We speculated that Mydgf might improve heart function via myocardial regeneration, which is essential for discovering the target to reverse heart failure.Methods: Two genetic mouse lines were used: global Mydgf knockout (Mydgf-KO) and Mydgf-EGFP mice. Two models of cardiac injury, apical resection was performed in neonatal and MI was performed in adult mice. Quantitative reverse transcription-polymerase chain reaction, western blot and flow cytometry were performed to study the protein expression. Immunofluorescence was performed to detect the proliferation of cardiomyocytes. Heart regeneration and cardiac function were evaluated by Masson's staining and echocardiography, respectively. RNA sequencing was employed to identify the key involved in Mydgf-induced cardiomyocyte proliferation. Mydgf recombinant protein injection was performed as a therapy for cardiac repair post MI in adult mice.Results: Mydgf expression could be significantly induced in neonatal mouse hearts after cardiac injury. Unexpectedly, we found that Mydgf was predominantly expressed by endothelial cells rather than macrophages in injured neonatal hearts. Mydgf deficiency impeded neonatal heart regeneration and injury-induced cardiomyocyte proliferation. Mydgf recombinant protein promoted primary mouse cardiomyocyte proliferation. Employing RNA sequencing and functional verification, we demonstrated that c-Myc/FoxM1 pathway mediated Mydgf-induced cardiomyocyte expansion. Mydgf recombinant protein improved cardiac function in adult mice after MI injury with inducing cardiomyocyte proliferation.Conclusion: Mydgf promotes cardiomyocyte proliferation by activating c-Myc/FoxM1 pathway and improves heart regeneration both in neonatal and adult mice after cardiac injury, providing a potential target to reverse cardiac remodeling and heart failure.

Myocardial infarction (MI) is a leading cause of death worldwide. During an MI, the contractile myocardium experiences reduced blood flow causing expansive cell death. Since adult mammalian hearts lack regenerative capacity, MI damage is permanent followed by heart failure. However, recent studies have demonstrated that the neonatal mouse heart exhibits transient regenerative potential within the first 7 days, suggesting a regulatory mechanism that prevents the adult from initiating a regenerative response to cardiac injury. The Hippo signaling pathway negatively regulates mammalian cardiac regeneration by inhibiting the activity of downstream effector YAP. However, YAP5SA, which completely bypasses Hippo pathway regulation, results in YAP hyper-transcriptional activity and heart overgrowth. In this work, we aim to investigate the TEAD-independent mechanisms of YAP in promoting cardiomyocyte (CM) division and renewal. We generated another YAP protein, which can not only skip Hippo pathway inhibition, but also lose the interaction with TEADs. We found that this modified YAP promoted CM proliferation and improved cardiac repair post MI injury. Our work will uncover novel functions of YAP and improve YAP therapeutics in myocardial infarction, finally benefiting human heart health.

MiRNA-210 transfected adult rat CMs show proliferation and reduced cell death in vitro. Cell cycle inhibitor APC is a target of miR-210. MiR-210 overexpressing (210-TG) mouse hearts show CMs cell cycle re-entry and survival post myocardial injury. 210-TG mice show significant neovascularization and angiogenic potential post myocardial infarction. 210-TG hearts show reduced infarct size following ischemic injury.

Arachidonate 5-lipoxygenase (ALOX5)-derived leukotrienes are primary signals of leukocyte activation and inflammation in response to ischemic cardiac injury (MI; myocardial infarction). Using risk-free male C57BL/6J and ALOX5-null mice (8-12 wk), we quantitated leukocytes and ALOX5-derived bioactive lipids of the infarcted left ventricle (LV) and spleen to measure the physiological inflammation and cardiac repair. Our results showed that ALOX5 endogenously generates specialized pro-resolving mediators (SPMs) that facilitate cardiac repair post-MI. Deficiency of ALOX5 leads to increase in cyclooxygenase gene expression, 6-keto prostaglandin F1α, and delayed neutrophil clearance with signs of unresolved inflammation post-MI. Consequently, ALOX5 deficiency impaired the resolution of inflammation and cardiac repair, including increased myocardium rupture post-MI in acute heart failure. On-time ALOX5 activation is critical for leukocyte clearance from the infarcted heart, indicating an essential role of ALOX5 in the resolution of inflammation. In addition, to balance the inflammatory responses, ALOX5 is also necessary for fibroblast signaling, as the ALOX5-deficient fibroblast are prone to fibroblast-to-myofibroblast differentiation leading to defective scar formation in post-MI cardiac repair. Consistent with these findings, ALOX5-null mice showed an overly inflammatory response, defective fibrotic signaling, and unresolved inflammation. These findings are indicative of a critical role of ALOX5 in myocardium healing, inflammation-resolution signaling, cardiac repair, and fibroblast pathophysiology.NEW & NOTEWORTHY Arachidonate 5-lipoxygenase (ALOX5) is critical in synthesizing specialized pro-resolving mediators that facilitate cardiac repair after cardiac injury. Thus, ALOX5 orchestrates the overlapping phases of inflammation and resolution to facilitate myocardium healing in cardiac repair postmyocardial infarction.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC EVICARE, Van Herk Foundation Introduction The use of cardiac progenitor cell (CPC)-derived small Extracellular Vesicles (sEVs) has shown great potential to stimulate cardiac repair post myocardial infarction. sEVs are released by cells and play a role in intercellular communication through transfer of their bioactive content, like RNAs and proteins. Increasing evidence indicates that sEVs present a heterogeneous mixture of vesicle populations, depicting different functional aspects, which hampers clinical translation. Studying sEV heterogeneity could provide new insights into contributing therapeutic mechanisms underlying sEV-mediated cardiac repair. Methods CPC-derived sEVs were purified by flow through chromatography followed by size-exclusion chromatography (SEC) and asymmetric flow field flow fractionation (A4F) for fractionation of different sEV-subfractions. Based on the differential expression of common sEV markers, different subpopulations were determined. sEV subpopulations were further characterized using western blot, nanoparticle tracking analysis, bicinchoninic acid protein assay, transmission electron microscopy and mass spectrometry (MS). Functional differences were studied using different cellular assays to determine AKT phosphorylation, wound healing, angiogenesis and proteasome activity. Results SEC or A4F were used to separate distinct subpopulations of CPC-derived sEVs with a different appearance, size and function. MS analysis confirmed the differences in proteomic expression levels of classical sEV markers, as well as annexins, rab proteins, integrins, histones and proteasomal proteins. Furthermore, differences in cellular components linked to their cellular origin were found. Exposure of recipient endothelial cells to sEV subpopulations demonstrated clear functional differences. Additionally, differences in proteolytic activity of the sEV subpopulations were identified. Conclusions SEC and AF4 allow for isolation and in-depth study of the functional heterogeneity of sEVs. In our study, we observed the presence of different subpopulations based on size, with a different composition and biological function. Increased knowledge of sEV heterogeneity will contribute to a better understanding of the mechanisms of action of sEVs and will improve translation to the clinic and potentially an off-the-shelf approach to stimulate cardiac repair.

SAMHD1 promotes cardiac repair post myocardial infarction by targeting NR4a1 to regulate macrophage metabolic reprogramming.

Hyperhomocysteinemia inhibited cardiac stem cell homing into the peri-infarcted area post myocardial infarction in rats