Role of Extracellular Vesicles in Cardiac Regeneration

Abstract

Heart failure remains a leading cause of morbidity and mortality worldwide. Despite advances in medical management and device-based therapies, there is no cure for the damaged heart. The traditional therapeutic options for patients with heart failure, such as drugs, surgeries, and transplantation, have limitations and risks, leading to the need for innovative novel therapies. Clinical and preclinical studies have shown that extracellular vesicles (EVs) secreted by transplanted cells are more effective than direct stem cell transfer in the mechanisms involved in cardiac regeneration following ischemia. EVs have gained increasing attention as potential mediators of cardiac repair and regeneration. Preclinical studies have demonstrated the regenerative effect of EVs from a variety of cardiac cell types, including cardiac progenitor cells, mesenchymal stem cells, and iPS cells. Upon EV administration, the functional capacity of the heart improved, myocardial hypertrophy reduced, and necrosis resulted in a lesser degree. This indicates that EVs’ ability to transport proteins, lipids, non-coding RNAs, and other biologically active factors plays a vital role in promoting cardiac restoration. At present, several clinical trials are exploring the therapeutic potential of EVs in heart regeneration approaches.

1. Introduction

Through the pioneering pediatric cardiac surgery in 1938 and the introduction of open-heart surgery on cardiopulmonary bypass in the 1950s mortality rates caused by congenital heart disease significantly decreased [1]. Worldwide, ischemia injury-related cardiovascular disease continues to be the primary cause of death. Many of the more complicated types of congenital heart disease, however, still cannot be physiologically repaired; instead, patients need surgical relief, which allows for survival but leaves behind considerable residual volume and pressure loads in the heart [2]. These patients are susceptible to heart failure over time, and some will eventually need a heart transplantation. Especially, Myocardial infarction (MI) is considered the most severe clinical sign of coronary artery disease (CAD) and one of the life-threatening coronary events associated causes the loss of 1 million cardiomyocytes. Myofibroblasts that produce collagen are activated by infarction (MI), which quickly sets off an innate immune response to remove dead or dying cells and restructure the extracellular environment [3].

The limited capacity of mature mammalian cardiomyocytes to multiply is a significant biological issue that restricts therapy options for patients with congenital heart disease as well as people with acquired heart disease. Since mature, mammalian cardiomyocytes do not re-enter the cell cycle to replace ischemic tissue, cardiac muscle injury is irreversible [4, 5]. Mammalian cardiomyocytes exit the cell cycle shortly after birth, even though cardiomyocyte proliferation is responsible for fetal heart growth. There is demonstrable, if modest, cardiomyocyte turnover in adult cardiomyocyte turnover rate is 1% per year at age 20 and rapidly declines with aging [6].

There are few viable therapeutic options that effectively reverse cardiac damage and restore heart function, even though pharmacological medicines, such as statins and anti-hypertensive medications, have improved the management of coronary vascular disease and its associated symptoms. The need for more efficient methods to enhance heart function grows as the population continues to age.

The use of stem cells in therapy holds promise for the treatment of vascular disorders, particularly due to our understanding of the mechanisms underlying stem cell activation, homing, and differentiation during vascular remodeling and repair.

Adult stem cells (ASC) have been suggested as a treatment for the infarcted heart for more than 20 years and ASC therapy for the heart has attracted a lot of attention in the clinical and basic scientific fields over the past decade [1]. The bulk of animal research and early human investigations using ASCs including Hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), BM-derived mononuclear cells (BM-MNCs), and Cardiac stem/progenitor cells (CPC) therapy after Acute MI have shown an improvement in heart function.

Among these, bone marrow-derived MSCs (BM-MSCs) and cardiac progenitor cells (CPCs), have received attention, due to their unique properties. MSCs are CPCs allow both autologous and allogeneic transplantation. They can differentiate into endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and cardiomyocytes. However, injection of BM-MSCs [2] and CPCs [3] into the infarcted murine hearts has improved left ventricular ejection fraction (LVEF), contractility, angiogenesis, and decreased infarct size in pre-clinical investigations [4, 5]. These cells have shown the ability to differentiate into vascular endothelial cells and cardiomyocytes in vitro. However, according to pre-clinical or clinical studies, there is no exact conclusion about the differentiation of MSCs and CPCs into cardiac cell types in transplanted hearts [6]. However, further research failed to replicate these results in bigger animal experiments, revealing poor engraftment of the injected cells and a danger of tumor growth when employing pluripotent stem cells. Despite earlier findings that promised promising results. Additionally, there has been a wave of pessimism in the clinical community due to the potential immunogenic reaction linked to allogeneic and xenogeneic stem cell transplantation. Additionally, research has consistently demonstrated that BM-MSCs [7] and CPCs [8] transplanted in patients engraft successfully or do not survive past 3 weeks post-injection. These results suggested that differentiation following transplantation cannot be the key mechanism that causes the marked improvements in cardiac outcomes. Therefore, there is a therapeutic need to find a novel medication that gets around these constraints while still significantly enhancing heart function.

The “paracrine hypothesis” postulates an alternate process involving the secretion of soluble paracrine substances [4]. Extracellular vesicles (EVs) have attracted much attention as major players of paracrine systems.

Cell-to-cell communication can be carried out through direct contact or through secreted signaling molecules. The mechanism of intercellular communication is based on the release and uptake of membrane-bound vesicles, termed EVs. EVs are ranging in size from 30 nm to 10 μM. EVs are heterogeneous populations and can be broadly categorized based on size: apoptotic vesicles which are bigger than 1000 nm in diameter; microvesicles ranging from 100 to 1000 nm; and exosomes ranging between 30 and 100 nm. Exosomes are the subtypes of EVs and they are considered as smallest EVs. However, generally in a few studies it is suggested that the therapeutic EVs are exosomes. Due to this reason, we will use EVs as an “umbrella term” throughout this chapter. EVs are released from the endosomal compartments of living cells and carry cargo such as lipids, proteins, messenger RNA (mRNA), micro-RNA (miRNA), and long non-coding RNAs (lncRNAs) that participate in intercellular communication. They are taken into the target cell by endocytosis, fusion with the plasma membrane, or binding to receptors on the cell membrane [9]. EVs are released from many cell types and can be found in blood, urea, and serum in the body and the culture medium (media) in cell culture studies. The EV membrane-specific proteins such as CD63, CD81, Alix, and Tsg101 are used for characterization and experimental verification.

An increasing amount of empirical data substantiates the proposition that paracrine mechanisms, facilitated by substances produced by ASCs, are integral to the observed reparative process following stem cell mobilization or injection into hearts afflicted with infarction. Research has demonstrated that ADSs specifically MSCs and CPCs, have the capability to generate and release a diverse array of cytokines, chemokines, and growth factors. These bioactive molecules hold promise for their potential involvement in the process of heart repair.

2. Paracrine hypothesis

At the beginning of the story, the main idea was that transplanted stem cells would engraft, differentiate into cardiac cell types, and replace damaged cardiac tissues. However, no therapeutically important degree of transplanted ASC engraftment or differentiation has yet been shown through experimental or clinical studies. Instead, they show that transplanted cells release substances that lessen tissue damage and/or improve tissue repair.

In general, stem cell transplantation has demonstrated a modest though consistent enhancement in cardiac function in preclinical models of myocardial infarction [10]. The initiation of clinical cell treatment trials in patients following acute myocardial infarction (MI) occurred expeditiously. The utilization of unfractionated autologous bone marrow mononuclear cells was largely observed in randomized clinical trials. The trials demonstrated a limited enhancement in heart function among individuals who underwent cell treatment [11, 12]. It is noteworthy to add that a phase II clinical experiment using the intracoronary infusion of cardiac-derived stem cells exhibited a notable enhancement in cardiac function among patients with single ventricle physiology [13].

Previously, it was thought that the injected cells had the power to generate new cardiac tissue. However, there is increasing evidence indicating that these cells really exert their effects indirectly by releasing paracrine substances that stimulate internal systems of therapeutic value [1, 14]. Recent studies have indicated a connection between EVs and their role as a primary element in paracrine signaling, which regulates the beneficial effects of stem cells in a manner that does not involve the transplantation of complete cells, thus avoiding associated challenges and drawbacks. In light of numerous pre-clinical and clinical research, it is demonstrated that adult stem cell therapy has beneficial benefits because of anti-apoptotic, immunomodulatory, and proangiogenic paracrine molecules that are released from cells.

EVs are considered as the major and active component of the paracrine secretion. Several recent studies have demonstrated the efficacy of utilizing EVs obtained from several types of stem cells, as a potential therapeutic approach to enhance heart function [15, 16, 17]. Therefore, it is believed that stem cells do not persist for an extended duration, but instead secrete paracrine factors that promote intrinsic myocardial repair mechanisms, including heightened cardiomyocyte cell cycle activity, neovascularization, and augmented involvement of stem cells in myocardial repair (Figure 1).

Figure 1.

The diagram represents the local and distal connections of the heart mediated by EVs. Various cardiac cell types release EVs that have the potential to exert autocrine effects on the originating cell. EVs utilize intercellular communication through the transfer of proteins, lipids, and nucleic acids between cells. This transfer occurs through mechanisms such as endocytosis, membrane fusion, or gap junction-mediated transfer. The abbreviation CM refers to cardiomyocyte, CSC stands for cardiac stem cell, and EV represents EVs. (adapted from Sahoo et al. [18]).

The paracrine idea was supported by many clinical and pre-clinical studies showing how human CPC-derived exosomes can recapitulate in large part the advantages of stem-cell therapy and improve heart function following myocardial infarction.

This chapter elucidates the therapeutic potential of EVs formed from stem cells, in conjunction with therapies based on stem cells.

3. Therapeutic potential of stem cell-derived EVs

3.1 Cardiac stem/progenitor cell-derived EVs

The replacement of cardiomyocytes that have undergone cell death is not yet achievable by existing therapeutic interventions, leading to predominantly irreversible impairment of cardiac function. The use of stem cells has brought a new potential in the treatment of heart failure. To promote the regeneration of myocardial cells and vascular cells, hence enhancing cardiac function, potential strategies may involve the transplantation of pluripotent/adult stem cells into the injured area after area myocardial infarction. Before a decade ago, it was thought that the adult mammalian heart was a post-mitotic organ which has not the self-renewal ability.

The discovery of cardiac stem and progenitor cells, which are found in the heart and in extra-cardiac regions, has ushered in a new field of study centered on the application of endogenous cellular regeneration mechanisms to repair an injured heart. Nowadays it is widely accepted that the adult heart can still undergo cardiomyocyte turnover, possibly from resident cardiac stem/progenitor cells e(CPCs), according to current research. Islet 1+, Sca-1+, c-kit+, and cardiospheres are a few resident CPC populations that have all been found to support cardiac repair to varied degrees [19]. The identification of resident cardiac stem cells with the potential to differentiate into cardiac cell lineages was described [20, 21]. Following this discovery, it has been demonstrated through genetic destiny mapping studies that CPCs have a role in adult mammalian cardiomyocyte replacement after injury [22, 23]. In the hearts of numerous animals, including rats, dogs, pigs, and humans, endogenous CPC populations have also been discovered and isolated in a large number of additional studies. According to this research, CPCs can differentiate into a variety of cell types, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells [24, 25].

CPCs are a variety of cells located in the atria, ventricles, epicardium, and pericardium of the heart. According to existing theories, CPCs are considered to remain quiescent and have minimal effects on cardiomyocyte renewal under physiologically normal circumstances. CPCs, on the other hand, can be triggered following injury and may differentiate into cardiomyocytes or vascular cells. Resident CPCs are a heterogeneous population. In the embryonic and adult heart, CPC populations have also been characterized by membrane markers such as c-Kit, Sca-1, Abcg-2, CD90, and transcription factors such as Isl-1, Nkx2.5, MEF2C, and GATA4 [20, 26].

The Cardiospheres/cardiosphere-derived cell (CDC) population is another CPC group that has been well characterized. CDCs are produced from atrial/ventricular biopsy tissue cultures and are a mixture of stromal, mesenchymal, and progenitor cells [27]. Due to the clonogenic and multilineage capability of CDCs in vitro, pre-clinical investigations have also shown that CDC transplantation is safe and effective. Patients diagnosed with acute MI were randomly selected to receive CDCs via intracoronary infusion autologously. CDCs as part of the Cardiosphere-derived Autologous Stem Cells to Reverse Ventricular Dysfunction (CADUCEUS)- phase I research. After six months, there was no difference in the LV ejection fraction in this clinical trial. However, the group that was injected CDC considerable reduction in infarct size was observed. Patients receiving CDCs demonstrated decreased scar mass, increased mass of the viable heart, and enhanced local contractility [28].

Through both direct and indirect processes, CPC implantation in a damaged heart may result in myocardial healing. The cardiac stem/progenitor cells have been shown to possess transdifferentiation capability in the heart, as evidenced by data obtained from animal experiments. However, researchers have seen a limited presence of the transplanted cells in the heart after the initial few days of transplantation. Recent evidence suggests that indirect mechanisms may be responsible for the beneficial effects of CPC transplantation on a damaged heart. It was demonstrated that rather than direct differentiation into cardiomyocytes and vascular cells, releasing paracrine factors that cause existing cardiomyocytes to proliferate and hyperplasia, causing local endogenous CPCs to differentiate, and fusing transplanted cells with cardiomyocytes may cause an improved neovascularization and favorable alterations in the cardiac scar [4, 29].

It was published that EVs derived from human CPCs reduced apoptosis triggered by serum deprivation in neonatal mouse HL-1 cardiomyocytic cell lines [30]. Using human umbilical vein endothelial cells (HUVECs) is a very common method to sign an angiogenic activity. The angiogenic activity of CPCs was also demonstrated while promoting tube formation in HUVECs.

In the same studies, the efficacy of EV transfer was also demonstrated in vivo. The administration of human EVs released by CPCs into the region around the infarcted area of acute myocardial infarcted (MI) rats resulted in a decrease in cardiomyocyte apoptosis and scar formation while increasing the density of blood vessels in the infarcted area. This outcome also causes an improvement in ventricular function [31].

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression by exerting post-transcriptional repression. The interchange of genetic material between cells through EVs has been recognized as a significant route for the transfer of mRNAs and miRNAs for the therapeutic benefit of EVs.

CPC-derived EVs are enriched with certain miRNAs, including miR-132,miR-210, and miR-146a-3p [31]. miR-210 and miR-132 involve invascular remodeling and anti-apoptosis and the expression of miR-132 mediates the endothelial tube development. The activation of miR-146 leads to the induction of myocardial inflammation and dysfunction in cardiomyocytes through intercellular communication [32].

Two completed phase I clinical trials have shown that CDCs can improve cardiac function in patients with heart failure by reducing scarring and attenuating ventricular remodeling [33]. At 1-year follow-up, 4 patients receiving CDCs showed continued improvement in cardiac function despite only getting one injection at the beginning of the research. As a result of this discovery, it was postulated that transplanted CDCs largely extend their beneficial effects by secreting paracrine substances such as exosomes at the site of injury, resulting in short-term cardioprotection and long-term activation of endogenous cardiac healing. The study by Gallet et al. described the efficacy and safety of CDC-exosomes in treating acute and chronic myocardial damage in big animals [34]. The authors set out to find out whether human CDC exosomes display comparable characteristics when tested in swine with myocardial damage because CDCs have previously been found to be slightly immunogenic with no symptoms of systemic immune response or harm [33].

The application of hypoxia preconditioning, namely subjecting stem cells to low oxygen levels, has been demonstrated to boost the viability of transplanted stem cells and augment their therapeutic efficacy in cardiac repair [31]. Hypoxia preconditioning has the ability to promote the survival, migration, and angiogenesis of bone marrow-derived cells, resulting in an improvement in their therapeutic effectiveness [35].

The last studies demonstrated with strong evidence that the release of anti-apoptotic exosomes from explant-derived cardiac progenitor cells (CPCs) can be augmented by microenvironmental stimuli, such as hypoxia [31, 33].

Nevertheless, the investigation into the regulatory influence of traditional cardiovascular medications on the release of exosomes produced from human cardiac progenitor cells (CPCs) was elusive. Given the previous findings that the enrichment of plasma exosomes derived from transplanted cardiac progenitor cells provided protection to the ischemic myocardium, it was postulated that comparable outcomes could potentially be achieved through pharmacological stimulation of cardiac-resident progenitor cells.

Importantly microarray analysis demonstrated that 11 miRNAs were significantly upregulated in exosomes derived from CPCs cultured under hypoxic circumstances compared to exosomes derived from cells cultured under normoxic settings. These microRNAs exhibit a covariation with angiogenic and antifibrotic responses. The transplantation of the exosomes produced from CPCs exposed to a 12-hour period of low oxygen levels (hypoxic) resulted in enhanced immediate and long-term performance, while also suppressing the development of fibrosis in ischemic heart [36].

The administration of conventional drugs such as ticagrelor to human cardiac-derived mesenchymal progenitor cells also enhances the level of exosome release. These exosomes are enriched with anti-apoptotic HSP70 [37]. These pre-clinical outcomes bring the potential to contribute to the advancement of new pharmacological interventions that are clinically significant for exosome/EV-based therapies.

3.2 Mesenchymal stem cell-derived EVs

Smooth muscle cells, osteoblasts, chondrocytes, and adipocytes are just a few of the different cell types that mesenchymal stem cells can differentiate into. Mesenchymal stem cells (MSCs) are subsets of stromal cells and were first identified by Friedenstein in 1970 [38]. Beyond bone marrow, MSCs are found in a variety of tissues. Due to their unique characteristics, such as their capacity to develop into cardiovascular cells, immunomodulatory properties, and antifibrotic activities, MSCs have an important role in the treatment of cardiovascular disease (CVD).

The release of angiogenesis and arteriogenesis factors, according to some research groups, is the primary mechanism by which transplanted bone marrow-MSCs (BMSCs). In rat models of MI stimulate heart healing and angiogenesis in models of cardiac damage significantly increasing vascular density (80%) and decreasing collagen content (33%). The transplanted cells had the biggest inhibitory effect on the loss of heart function and enhanced vascular healing [39, 40, 41]. The efficacy of BMSC was also reported in big animals such as pigs. Amado et al. demonstrated that BMCS cells that were transplanted had been pre prepared from an allogeneic donor, and their successful integration without rejection represents a significant practical advancement in facilitating the widespread use of this therapeutic approach [42].

At the moment, bone marrow, adipose tissue, and cord blood are the primary sources of MSCs employed in clinical trials [43]. The most often employed cells in the therapy of CVD are adult allogeneic MSCs produced from bone marrow. The first clinical trial to employ intravenous allogeneic hMSC in MI was carried out in the United States in 2005 by Joshua Hare and associates (NCT00114452) [44]. In this study, MSCs were used in a clinical trial that was randomized and parallel-assigned to treat acute MI (heart attack). Phase I/II clinical study of the intramyocardial delivery of allogeneic human umbilical cord mesenchymal stem cells (HUC-MSCs) to patients with chronic ischemic cardiomyopathy was also reported by Ankara University in 2015 [45].

According to the research mentioned above, MSC therapy is safe and can enhance myocardial perfusion following MI. The clinical trials demonstrate the MSC transplantation’s good safety profile and the absence of tumor formation that has been documented. However, there are certain possible dangers associated with the systematic administration of MSCs, including embolism and inflammation. Although researchers in CVDs have carried out several clinical trials, this form of trial is still in its very early stages.

Although researchers have documented the differentiation of MSCs into cardiomyocytes, it is commonly acknowledged that the main impact of MSCs in the therapy of CVDs depends on the paracrine action.

Numerous further investigations have demonstrated that MSCs take part in immunological modulation via paracrine mechanisms. Transplantation dramatically improved the cardiac function of MI mice by lowering the production of IL-1, IL-6, and TNF- alpha as well as the death of myocardial cells [46].

According to recent studies [4, 47], paracrine substances released by MSCs may be a mediator of some of these reparative effects. Numerous studies have found that MSCs release cytokines, chemokines, and growth factors that may be used to potentially heal damaged cardiac tissue, mostly through the formation and regeneration of cardiac and vascular tissue. This is evidence in support of the paracrine concept. According to Pittenger and Martin [48], this paracrine concept may offer an alternative to employing MSCs that are not cell-based for the treatment of cardiovascular disease. Contrary to cell-based therapies, non-cell therapies are considered as simpler and safer.

Using MSCs to treat a rat model of MI was found to improve cardiac function by lowering the number of CD68-positive inflammatory cells and monocyte chemotactic protein-1 (MCP-1) in the myocardium [49]. Studies demonstrate that MSCs that are transplanted into the vicinity of MI increase left ventricular remodeling and function by inhibiting miR-155-mediated profibrotic signaling and releasing HGF through direct cell contact [50]. Experimental underpinnings of the extensive clinical bone marrow-derived progenitor/stem cell transplantation trials that are currently being conducted in patients with myocardial infarction [2, 51] as these trials were largely founded on the presumption that bone marrow-derived cells produce significant amounts of cardiomyocytes through transdifferentiation.

MSCs are involved in paracrine pathways that promote angiogenesis. Ju et al. demonstrated that MSC-secreted exosomes enhanced capillary density and cardiomyocyte proliferation [52]. It was also shown that exosomes from hypoxia induced-MSCs induce angiogenesis both in in vitro and in vivo models through regulating HIF-1 [53, 54].

MSC-derived exosomes have also a cardioprotective impact on CPCs. The researchers observed that the proliferation, migration, and development of angiotubes in CSCs were enhanced after the treatment of CPCs with MSC-derived exosomes. In an experimental model of myocardial infarction in rats, the administration of mesenchymal stem cell-derived exosomes that were preconditioned with cardiac stem cells (CSCs) resulted in notable improvements in capillary density, reduction in cardiac fibrosis, and restoration of cardiac function.

The examination of miRNA profiling showed that a specific group of miRNAs exhibited significant alterations in CSCs following treatment with MSC-Exo. In an experimental model of myocardial infarction in rats, it was observed that CPCs that were preconditioned with exosomes derived from MSCs exhibited notable improvements in terms of engraftment, survival rates, capillary density, reduction in cardiac fibrosis, and restoration of long-term heart function [55].

As we mentioned above research conducted in pre-clinical settings has demonstrated that the application of hypoxia preconditioning could augment the therapeutic capacity of stem cells for the purpose of cardiac repair. Simultaneously, the administration of MSC exosomes has the capacity to modify the expression levels of microRNAs (miRNAs). It was shown that miR-210 and miR-744 exhibited an elevation in exosomal levels in response to hypoxia. miR-210 was selected as the principal factor in our investigation of myocardial protection providing protection to cells from harm through paracrine signaling. Moreover, some investigations have shown a correlation between exosomal-miR-210 and its role in safeguarding against ischemia injury. MSCs release exosomes containing a high concentration of miRNA-210 under hypoxic conditions [56].

Under conditions resembling peripheral arterial disease, MSCs lead to an upregulation of various angiogenic proteins which are encapsulated within exosomes and secreted by the MSCs. The released exosomes then stimulate angiogenesis in endothelial cells. The proteome of MSCs saw notable alterations when exposed to an environment resembling peripheral arterial disease. The examination of the angiogenesis interactome of proteins found in MSCs demonstrated that the strongest clustering of interactions with signaling proteins occurred with platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and NFkB nodes, suggesting proangiogenic capabilities of MSCs [57].

3.3 The significance of EVs released by intracardiac cells

The heart has not been traditionally considered as a secretory organ for a long time. Recent research indicated that myocardial tissue, including cardiomyocytes, endothelial cells (ECs), fibroblasts (Fbs), and cardiac progenitor cells (CPCs), releases EVs that play a significant role in facilitating intercellular communication within the cardiac system.

Communication between cardiac muscle cells and ECs includes EV-mediated pathways. The overexpression of HIF-1α in cardiomyocytes from newborn piglets exposed to hypoxia was found to result in the induction of Hsp20 which exhibits angiogenic properties by promoting the upregulation of vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells (ECs). Furthermore, it has been demonstrated that exosomes enriched with miR-126 and miR-210, derived from endothelial cells exposed to hypoxia, enhance the resistance of cardiac progenitor cells (CPCs) to hypoxic stress by activating the PI3K/Akt pathway and other pathways associated with cell survival [58, 59].

Cardiometabolic drugs also exert their effect on EVs secreted by ventricular cardiomyocytes under hyperglycemia. Ticagrelor treatment modulates the EVs released from H9c2 cells via reducing enhanced ROS production, ER stress, and autophagy [60]. The investigation of the intricate signaling pathways involved in the activities of cardiometabolic drugs can offer a potentially advantageous pharmacological approach for safeguarding the heart from pathogenic stimuli.

4. Engineered EV strategies in cardiac therapy

On the contrary to the therapeutic efficacy of EVs, there are also limitations in EV-based therapies such as targeting, tracking, bioactivity, and internalization of transferred EVs.

In the past decade, engineered EV-based techniques have been developed to deliver specialized cargos to specific targeted tissues and promote the stability of EVs. The new strategies in the modulation of EVs including surface engineering and cargo loading provide promising therapies. There are two types of strategies; endogenous and exogenous methods in engineered EV-based therapies [61]. In the endogenous method, modulation processes are completed in paternal cells such as overexpression of nucleic acid and protein in the cells before isolation. In the exogenous method, EVs are directly engineered with nucleic acids and targeting ligands using physical or chemical methods following isolation from cells.

EV-biodistribution is a very important factor during the evaluation of the parameters such as efficiency of targeted delivery, administration route, concentration of EVs delivered, dispersion of EVs in body fluids, and the source of the EV-secreting cell in therapy [62]. For imaging purposes, EVs can be labeled after the isolation (exogenous method) or paternal cells can be genetically modified to EVs express a reporter protein (endogenous method). Fluorescence, nuclear (single photon emission computed tomography (SPECT) or positron emission tomography (PET)), and luminescence imaging techniques are used in studies to monitor EVs in vivo.

Cell source is a very effective parameter in the biodistribution of EVs. It is also suggested that EVs from different cell sources demonstrate a high tendency to accumulate in to originated- tissue [63]. On the other hand, a variety of studies reported that intravenously injected EVs show a higher preference to accumulate in mononuclear phagocyte system (MPS) organs like spleen, lung, and liver. Regardless of the delivery route, EVs are absorbed and destroyed by macrophages very quickly in blood flow. These kinds of limitations led the researchers to develop the methodologies for targeted delivery of EVs for treating heart diseases. Studies also demonstrated that intramyocardial delivery is more effective compared to intracoronary and intraperitoneally administration indicating that the delivery route of injection influences the distribution of infused EVs.

One method to increase the stability of EVs in circulation is modifying the EV membrane with polyethylene glycol (PEG). Following PEG covering, nanobodies specific to the targeted tissue are conjugated to prepare nanobody-PEG-micelles. This method increases the circulation time while reducing their uptake by non-specific cells at the same time [64].

Modification of the EV membrane with specific proteins or peptides that can interact with specifically cellular receptors or extracellular matrix components expressed in the cardiovascular system is considered as very reliable strategy to improve cardiac tropism.

Exosomes derived from cardiosphere-derived cells (CDCs) were engineered with a Lentivirus to express exosomal membrane protein Lamp2b that is fused to a cardiomyocyte-specific peptide (CMP), WLSEAGPVVTVRALRGTGSW targeting ischemic myocardium. This strategy caused an increase in the uptake of exosomes by cardiomyocytes and enhanced retention of EVs compared to un-modified exosomes [65].

To improve the bioactivity of EVs, secreting cells can be modulated by different exogenous strategies. Through these strategies, especially miRNA profiling of EVs could be changed in a cardioprotective manner. For instance, under hypoxia CPCs release proregenerative exosomes as a response. Differentially regulated miRNAs were indicated compared to normoxic conditions including miRNA-15b, miRNA-103, miRNA-20a, miRNA-210, miRNA-199a, and miRNA-292. Exosomes from hypoxic CPCs were delivered to the heart following ischemia–reperfusion injury. It was validated that exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis compared to those cultured under normoxic conditions [36].

EV miRNA profiling also can be modulated by changing the culture medium or transfecting the paternal cells to upregulate/downregulate one (or more than one) specified miRNA expression level of released EVs to enhance and lead the cardioprotective effect of delivered EVs [66, 67].

Following intramyocardial injection of EVs which are isolated from miRNA-181a transfected-MSCs into mouse hearts improves heart functions compared to non-modulated EVs [67].

Another important engineering strategy is a post-isolation modification of EVs. This method provides EV loading to modulate the enrichment of EVs. Through changing protein, lipid, and especially miRNA material of EV after isolation, we could lead the targeting and delivery regardless of their cell of origin. There are different loading strategies that are approaching such as electro-poration, heat–shock/freeze–thaw procedures, sonication, or passive loading methods like incubating the EVs with interested drugs, or molecules [63].

For instance, EVs isolated CPCs enriched with miRNA-322 by electroporation and transferred to a mouse MI heart. This strategy causes an increase in angiogenesis and reduction of fibrotic area compared to the use of non-modified EVs [68].

All these strategies aim to enhance the efficiency of EV-based treatment for cardiovascular diseases. Hence, many studies are still required to use the advantage of EV engineering to improve the targeting, retention, and bioactivity abilities.

5. EVs as biomarkers in heart diseases

EVs carry information from cell-to-cell referring that they also respond to pathophysiological changes. It is well known that EVs are directly associated with physiological processes, such as thrombosis, apoptosis, inflammation, cell survival, endothelial dysfunction, and angiogenesis [69]. Especially, there is an increasing attention on plasma EVs as potential diagnostic/prognostic biomarkers for cardiovascular diseases.

Plasma EV proteins cystatin C, serpin C1, CD14, and serpin F2 levels are associated with an increased long-term major cardiovascular event risk after carotid endarterectomy suggesting cardiovascular risk markers [70].

Numerous studies reported that exosomal miRNAs such as miR320b, miR133a, miR143, miR150, miR155, miR214, miR223, and miR320b in circulation are diagnostic biomarkers for vascular inflammation and atherosclerosis [71].

In addition, exosomal miR208a, miR1, miR499-5p, and miR30a were identified as the early diagnostic markers of acute myocardial infarction [72].

6. Conclusions

To date, there has been significant attention focused on EVs due to their important properties, in relation to their potential for diagnostic and therapeutic applications in the treatment of cardiac diseases. According to preclinical findings, it is proposed that therapies utilizing EVs hold the potential to replace cell therapy in clinical settings.

The results indicate that the utilization of cardiac-derived stem cell therapy in patients is generally regarded as a safe approach. Nevertheless, the observed enhancements in cardiac function exhibit a somewhat modest and gradual pace of advancement. Furthermore, there is an ongoing debate surrounding the precise mechanism underlying the advantageous outcomes of stem cell therapy.

EVs demonstrate comparable advantages in stem cell transfer. Nevertheless, there remains a need to translate the results obtained from studies conducted on small animal models to a larger animal model that is clinically significant for investigating cardiac damage.

The therapeutic benefits of EVs in cardiac failures were very well reported in preclinical studies. However, due to the concerns about the off-target effects of EVs, the safety for use in human trials is required more pre-clinical studies.

Conflict of interest

The authors declare no conflict of interest.