Cardiac Progenitor Cell–Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis

Myocardial Regeneration via Progenitor Cell-Derived Exosomes

In the past 20 years, a variety of cell products has been evaluated in terms of their capacity to treat patients with acute myocardial infarction and chronic heart failure. Despite initial enthusiasm, therapeutic efficacy has overall been disappointing, and clinical application is costly and complex. Recently, a subset of small extracellular vesicles (EVs), commonly referred to as “exosomes,” was shown to confer cardioprotective and regenerative signals at a magnitude similar to that of their donor cells. The conceptual advantage is that they may be produced in industrial quantities and stored at the point-of-care for off-the-shelf application, ideally without eliciting a relevant recipient immune response or other adverse effects associated with viable cells. The body of evidence on beneficial exosome-mediated effects in animal models of heart diseases is rapidly growing. However, there is significant heterogeneity in terms of exosome source cells, isolation process, therapeutic dosage, and delivery mode. This review summarizes the current state of research on exosomes as experimental therapy of heart diseases and seeks to identify roadblocks that need to be overcome prior to clinical application

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction

<div><p>Aims</p><p>Hereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.</p><p>Methods and results</p><p>After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (<i>Eng</i>^+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4⁺ MNCs residing in the infarcted <i>Eng</i>^+/- hearts (<i>Eng</i>^+/- 73.17±12.67 vs. <i>Eng</i>^+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (<i>Eng</i>^+/- 46.60±9.33% vs. <i>Eng</i>^+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in <i>Eng</i>^+/- mice. An increased number of capillaries (<i>Eng</i>^+/- 61.63±1.43 vs. <i>Eng</i>^+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (<i>Eng</i>^+/- 11.88±0.63 vs. <i>Eng</i>^+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in <i>Eng</i>^+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. <i>Eng</i>^+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.</p><p>Conclusions</p><p>In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in <i>Eng</i>^+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.</p></div

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.ISSN:1757-651

Monocyte specific knock-out of endoglin does not recapitulate the <i>Eng</i>^<i>+/-</i> phenotype.

<p><b>(A)</b> Western blot analysis of endoglin protein expression in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages. A representative experiment is shown. <b>(B)</b> Quantification of the Western blots for endoglin protein in LysM-Cre-<i>Eng</i>^+/+, LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl cultured macrophages in two independent experiments(macrophage cultures from 3 individual mice of each genotype were pooled per western blot). <b>(C)</b> Kaplan-Meier survival curve of wild type (WT), LysM-Cre-<i>Eng</i>^fl/+ and LysM-Cre-<i>Eng</i>^fl/fl mice 28 days post-MI (n = 6–10). <b>(D)</b> Cardiac function in percentage ejection fraction (ï) 14 days post-MI. Cardiac function was measured by ultrasound in long axis view (n = 5–9). Data are shown as mean ± SEM, *P<0.05.</p

DPP4 inhibitor treatment reduces infarct size.

<p><b>(A)</b> Experimental protocol and treatment overview. At day 0, MI is induced and DPP4 inhibition is started (shown in green) till day 5 post-MI by intraperitoneal (i.p.) injection of DipA (treatment group) or distilled water (control group). Cardiac echography was performed at day 7 and 14 post-MI. <b>(B)</b> Histological analysis of the infarct size by Picrosirius red staining for collagen (n = 5–9). Transverse sections of left ventricle, photos taken at 1.0x magnification. Infarct area = dark pink, healthy myocardium = light pink, blood cells = yellow. <b>(C)</b> Quantification of Picrosirius red staining in left ventricle (LV). Mice were subjected to MI and treated with either distilled water or DipA from day 0 till day 5 by daily i.p. injection (n = 5–9). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

DPP4 inhibitor treatment of <i>Eng</i>^<i>+/-</i> mice restores homing of MNCs to injured myocardium at 4 days post-MI.

<p><b>(A)</b> Representative microscopy images of CXCR4 expression in the infarct border zone. White = surviving myocardium, black/grey area = infarcted myocardium. Photos taken at 30x magnification. Scale bar: 50μm. CXCR4 = red, cTnI = white, DAPI nuclear staining = blue. <b>(B)</b> Quantification of CXCR4 expressing cells in the infarct border zone (n = 6–8). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Quantification of MAC3 positive cells in the infarct border zone (n = 6–7). Data shown are mean ± SEM, *P<0.05. <b>(D)</b> Representative microscopy images of MAC3⁺/CD206^- (%M1) and MAC3⁺/CD206⁺ (%M2) expressing cells in the infarct border zone. Smaller panels: Top panel is the MAC3 signal, lower panel is the CD206 signal. Photos taken at 50x magnification. Scale bar: 20μm MAC3 = red, CD206 = green, DAPI = blue. <b>(E)</b> Quantification of the ratio of MAC3⁺/CD206^- (%M1) and MAC3⁺/CD206⁺ (%M2) expressing cells in the infarct border zone (n = 6–7). <b>(F)</b> Flow cytometric analysis of the macrophage population in the infarct area, ratio of inflammatory M1(Ly6G^-/CD11b⁺/Ly6C^high) versus regenerative M2 macrophages (Ly6G^-/CD11b⁺/Ly6C^low) (n = 3–6, non-parametric ANOVA testing). Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p

DipA treatment does not maintain improved cardiac function of <i>Eng</i>^<i>+/-</i> mice after MI.

<p><b>(A)</b> Experimental overview and percentage EF at 7 and 14 days post-MI of control treated mice, measured by ultrasound via left ventricle tracing (n = 5–9). Data shown are mean ± SEM, *P<0.05. <b>(B)</b> Percentage EF 7 and 14 days post-MI of DipA treated mice, measured by ultrasound via left ventricle tracing. Note that the control WT and <i>Eng</i>^<i>+/-</i> groups are the repeat of measurements used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189805#pone.0189805.g001" target="_blank">Fig 1D</a> (n = 9–11). Data shown are mean ± SEM, *P<0.05. <b>(C)</b> Long term treatment overview and Δï. DipA treatment (shown in green) up to 14 days post-MI and cardiac function with extended follow-up of 6 months (n = 5–11). Data depicted as ΔEF are the EF at the time point indicated on the x-axis compared to EF measured at day 7 post-MI. Cardiac function was measured by ultrasound via left ventricle tracing. DipA = DPP4 inhibitor Diprotin A, US = Ultrasound measurement. Control = MQ treated, DipA = Diprotin A treated group. Data shown are mean ± SEM, *P<0.05.</p