Exosomes are nanovesicles released from cells through exocytosis and are known mediators of proximal and distant cell-to-cell signaling. Still little is known and written about cardiac exosomes, although Gupta and Knowlton described exosomes containing HSP60 in 2007 [1]. It is now known that exosomes from cardiomyocytes can transfect other cells and that the metabolic milieu of the parental cell dictates the quality of exosomes released. So while their exact mediators remain unidentified, this, they induce, is what influences differential gene expression inside transfected cells. Future clinical use of exosomes in diagnosis, monitoring disease progress, and treatment is promising.

Their research suggests that cardiac progenitor cells (CPCs) are able to improve cardiac function after injury. The underlying mechanisms are indirect gene expression of transfected extracellular vesicles. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signaling mediators. Here, we report that EVs secreted by human CPCs are crucial for the production of cardio-protective agents [2].

CPCs on Nanofibers

Cardiac progenitor cells. Image courtesy of: Cell.com

Ischemic heart disease and heart failure are leading causes of morbidity and mortality worldwide. An extensively investigated approach for ischemic heart disease is cell transplantation. Multiple cell sources have been evaluated in both animal models and humans including bone marrow, adipose tissue, skeletal myoblasts, and cardiac progenitor cells (CPCs) [3]. Clinical trials of autologous BM-cell transplants in patients after acute myocardial infarction (MI) have provided mixed results, with an overall modest improvement in cardiac function [4]. Intramyocardial injection of CPCs isolated from adult hearts improved cardiac function in animal models of MI [5]. Although both direct cell differentiation and indirect mechanisms, such as secreted growth factors and cytokines, have been implicated in the therapeutic benefit, accumulating evidence suggests predominant roles of the paracrine secretion by CPCs. This concept is supported by the observation that CPC-conditioned medium (CM) protects cardiomyocytes against stress-induced apoptosis, while stimulating tube formation in endothelial cells in vitro [6]. This research has shown that cardiac regeneration in injured hearts may be possible, to some degree.

The mechanism of action is thought to occur through indirect cellular communication in extracellular vesicles (EVs) [7].

A key component of paracrine secretions in many cell types are EVs, particularly the exosome fraction. Exosomes are 40-90 nm-sized particles stored intracellularly in endosomal compartments, which are secreted when these structures fuse with the plasma membrane [8].

tetraspanin_structure

 

Figure 1. As can be inferred from their name, tetraspanins contain four transmembrane (TM) domains. They differ from other proteins with four TM domains by a shared overall structure of the extracellular and intracellular domains and by the presence of conserved amino acid residues. [Photo courtesy of: http://physiologyonline.physiology.org.]

Conventional surface markers of exosomes include the tetraspanin family members, CD9, CD63, and CD81 (seeFigure 1), among others. Exosomes carry proteins, lipids, and nucleic acids including DNA, mRNAs, and microRNAs (miRNAs), which they can shuttle to recipient cells, even at a distance. miRNAs are 18-25 nucleotide, non-coding RNAs that regulate gene expression through post-transcriptional repression.

Transfer of mRNAs and miRNAs through exosomes has emerged as a crucial mechanism of genetic exchange between cells [9,10]. Non-exosomal EVs arise by distinct mechanisms including direct budding from the plasma membrane. While non-exosomal EVs are typically larger than exosomes, exosome purification remains a challenging task. As a result, exosome preparations used in many studies have had limited purity [11].

Exosomes were identified as the active component of the pro-angiogenic paracrine pathway of bone marrow CD34+stem cells [12]. Moreover, exosomes secreted by mesenchyme stem cells (MSCs) and mouse CPCs, both mitigated tissue damage and helped remodel heart ventricules in animal models of myocardial ischemia and reperfusion injury [13,14]. However, exosomes secreted by human CPCs have yet to be fully characterized by researchers. Using transmission electron microscopy, researchers provided structural evidence that exosomes and EVs are actually secreted by human CPCs [15]. They also report that EVs are the active component of the paracrine secretion by human CPCs. Transmission was tracked using an electron microscope and nanoparticle analysis, which showed most EV secretion by CPCs to be 30-90 nm in diameter. To them, this confirmed the presence of exosomes (although smaller and larger vesicles were also present). More importantly, the purified particles expressed conventional exosome markers, including CD63. These results indicate that exosomes are the predominant fraction of the particles purified from CM-CPC.

mirna_expression

 

Figure 2. (G) Relative expression of miR-210 and miR-132 in EV-CPC compared with EV-F (mean ± SEM; n = 6 experiments; P < 0.05 using the parametric Tukey Kramer multiple comparison test). (H) Intracellular concentrations of miR-210 and miR-132 in HL-1 cells exposed to EV-CPC (100 µg of total protein) for the indicated periods of time (upper panel; n = 3 experiments). Lower panels: Dose–response studies (measurements at 48 h;n= 3 experiments). [Barile]. Image courtesy of Oxford Journals.

These EVs also mitigated apoptosis, triggered by serum deprivation in the neonatal mouse HL-1 cardiomyocytic cell line, while stimulating tube formation in human umbilical vein endothelial cells (HUVECs), one indication of its angiogenic activity. In vivo, injection of EVs secreted by CPCs into the infarct border zone reduced cardiomyocyte apoptosis and scar, increased both viable mass in the infarct area and blood vessel density, and prevented the early impairment of ventricular function in a rat model of acute MI. EVs secreted by normal human dermal fibroblasts (NHDFs) lacked these beneficial effects, supporting the concept that functional activities of EVs depend on the parent cell [16]. Comparing the miRNA transcriptional profile of EVs secreted by CPCs with that of EVs secreted by NHDFs identified several miRNAs as being particularly enriched in the former, including miR-210, miR-132, and miR-146a-3p.

Because cardioprotective roles of miR-210 and a role for miR-132 in vascular remodelling has previously been reported [17,18], Barile and co-workers instead focused on the functional activities of these miRNAs. Their results show that miR-210 and miR-132 appear to have protective properties on HL-1 cardiomyocytic cells, guarding them against apoptosis. In addition, miR-132 promoted the formation of endothelial tube. These results suggest that EVs secreted by CPCs may imbue cardiac protection and the underlying mechanism may possibly involve miRNAs.

 

SOURCES

[1] Gupta S, Knowlton AA. HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway. Am J Physiol Heart Circ Physiol. June 2007. 292(6):H3052-6.

[2,3] Barile L, Lionetti V, Cervio E, Matteucci M. Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction. Oxford Journals. Cardiovascular Research. July 11, 2014. <103 (4):530-541.doi: 10.1093/cvr/cvu167>.

[3,5,6] 5,6 Chimenti I, Smith RR, Li TS, Gerstenblith G, Messina E.Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice. Circ Res2010;106:971-980

[4] Marbán E, Malliaras K. Mixed results for bone marrow derived cell therapy for ischemic heart disease. JAMA 2012;308:2405-2406.

[7] Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LE. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet 2012;379:895-904.

[8] Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics 2010;73:1907-1920.

[9] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654-659.

[10] Stoorvogel W, Functional transfer of microRNA by exosomes. Blood. 2012;119:646-648.

[11] Bobrie A, Colombo M, Krumeich S, Raposo G, Thery C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles2012;1:18397.

[12] Sahoo S, Klychko E, Thorne T, Misener S, Schultz KM. Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity. Circ Res2011;109:724-728.

[13] Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res2010;4:214-222.

[14] Chen L, Wang Y, Pan Y, Zhang L, Shen C, Qin G. Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury. Biochem Biophys Res Commun2013;431:566-571.

[15] Barile L, Gherghiceanu M, Popescu LM, Moccetti T, Vassalli G. Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres. J Biomed Biotechnol 2012;2012:354605

[16] Sahoo S, Klychko E, Thorne T, Misener S, Schultz KM, Millay M, Ito A, Liu T, Kamide C, Agrawal H, Perlman H, Qin G, Kishore R, Losordo DW. Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity. Circ Res 2011;109:724-728

[17] Hu S, Huang M, Li Z, Jia F, Ghosh Z, Lijkwan MA, Fasanaro P, Sun N, Wang X, Martelli F, Robbins RC, Wu JC.MicroRNA-210 as a novel therapy for treatment of ischemic heart disease. Circulation 2010;122:S124-S131.

[18] Katare R, Riu F, Mitchell K, Gubernator M, Campagnolo P, Cui Y, Fortunato O, Avolio E, Cesselli D, Beltrami AP, Angelini G, Emanueli C, Madeddu P. Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Novelty and significance. Circ Res 2011;109:894-906.