Challenges and future scope of exosomes in the treatment of cardiovascular diseases

Abstract

Exosomes are nano-sized vesicles that carry biologically diverse molecules for intercellular communication. Researchers have been trying to engineer exosomes for therapeutic purposes using different approaches to deliver biologically active molecules to the various target cells efficiently. Recent technological advances may modify the biodistribution and pharmacokinetics of exosomes to meet scientific needs with respect to specific diseases. However, it is essential to determine the exosome’s optimal dosage and potential side effects before its clinical use. Significant breakthroughs have been made in the past decades concerning exosome labeling and imaging techniques. These tools provide in situ monitoring of exosome biodistribution and pharmacokinetics and pinpoint targetability. However, because exosomes are nanometers in size and significantly vary in contents, a deeper understanding is required to ensure accurate monitoring before it can occur in clinical settings. Different research groups have established different approaches to elucidate the roles of exosomes and visualize their spatial properties. This review covers current and emerging strategies for in vivo and in vitro exosome imaging and tracking for potential studies.

Graphical Abstract

Exosome, a nanosized extracellular vesicle, is secreted in various biological fluids from different cells/organs. The diagram shows the different strategies to isolate exosomes with purity. The steps involved in exosome isolation are purification and characterization using different approaches. In addition, exosomes can be engineered for better targeting and efficient therapeutic benefits, such as hydrogel, protein modification, ChIP, and molecular imprinting. The engineered exosomes can be biocompatible and easily transport drugs without adverse effects.

1. Introduction:

Various studies have identified the role of exosomes in cross-communication within different organs and tissues. Under different physiological conditions, the heart cells communicate with other organs through exosomes. Previous studies have shown that exosomes carry several molecular signatures, including lipids, proteins, mRNAs, and miRNAs (Vader et al., 2014; Ranjan et al., 2019; Ranjan et al., 2021). Exosomes are secreted selectively through cargo sorting mechanisms from almost all cell types in response to various biological stimuli and shuttle proteins, lipids, or nucleic acids to the recipient cells (Momen-Heravi et al., 2018; van Niel et al., 2018; de Jong et al., 2020). For cargo delivery, exosomes are either fused with different receptors (such as ICAMs, galectin, annexin V) on the recipient cells or endocytosed by the recipient cells (Christianson et al., 2013; Tian et al., 2014; Gonda et al., 2019; Fuentes et al., 2020). Several studies have described the role of exosomes in cardiac pathophysiology, such as apoptosis, angiogenesis, inflammation, hypertrophy, and fibrosis. The role of miRNAs carried by exosomal cargo has been extensively studied in cardiac pathology (Saludas et al., 2021). In apoptosis, cell senses stress signals and induces cell death by activating caspases. The role of exosomes has been implicated in regulating the apoptotic progression. For example, upregulating miR-423-3p in exosomes exerts cardio-protection and reduces apoptosis in H9c2 cells by downregulating RAP2C (Luo et al., 2019). Another in vitro study showed that cardiac progenitor cells-derived exosomes secreted miR210 and miR132 and inhibited apoptosis in cardiomyocyte and enhanced angiogenesis in cardiac endothelial cells respectively (Barile et al., 2014). Cardiac hypertrophy is an adoptive response to hemodynamic stress and is characterized by ventricular wall thickening and reduced ejection fraction. Elevated levels of miR-27a-, miR-28-3p-, and miR-34 have been seen in cardiac fibroblasts under the influence of TNFα, which promotes hypertrophy after being taken up by cardiomyocytes (Tian et al., 2018a). Myocardial ischemia is a condition of decreased blood flow to the heart and ischemic cell death that can lead to a multiphase reparative response with fibrotic scarring at the site of damaged tissue. Interestingly, M1-macrophages shuttle pro-inflammatory miR-155 to the endothelial cells via exosomes, thereby reducing its angiogenic potential and aggravating myocardial injury following myocardial ischemia (Liu et al., 2020). Previously, we have also identified that upregulated miR-200a-3p from activated fibroblast-derived exosomes induce cardiac endothelial cell dysfunction (Ranjan et al., 2021). Similarly, several other miRNAs are described in different diseases, including CVDs. Therefore, researchers have recently shown interest in exploiting exosomes for therapeutic use.

During infection or injury, the heart muscle naturally swells under the influence of chemicals secreted from the white blood cells. It is evident from the existing literature that a variety of cells secretes exosomes within the innate and adaptive immune systems, such as neutrophils, mast cells, NK cells, monocytes, macrophages, B cells, and T cells (Lindenbergh & Stoorvogel, 2018). Transient inflammation triggered by immune cells is the critical cardioprotective event in MI that clears dead cells from the infarct area. In this process, exosomal vesicles interact with infiltrating immune cells and influence immune cell polarization and cytokine secretion, thus exert cardioprotection (Wen et al., 2021). For example, exosomes secreted by cardiosphere-derived stem cells transduce miR-181b, induce macrophage polarization, and promote cardiovascular repair (Nahrendorf et al., 2010; Frangogiannis, 2012; de Couto et al., 2017). Exosomes secreted by M2 macrophages deliver miR-148a and attenuate MI/R injury through down-regulating TXNIP (Dai et al., 2020).

In contrast, M1 macrophage-derived exosomes deliver miR-155 and exacerbate myocardial injury (Liu et al., 2020). Recent studies advocate that those beneficial effects of stem cells are mainly due to their paracrine signaling via exosomes (Segers & Lee, 2008; Singla, 2016). Mesenchymal stem cell-derived exosomes provide cardio-protection against myocardial infarction through several mechanisms that might target apoptosis, inflammation, remodeling, and neovascularization (Golpanian et al., 2016; Cui et al., 2017; Joo et al., 2020). Ribeiro-Rodrigues et al. showed that cardiomyocyte-derived exosomes exert proangiogenic effects through exosomal miR-143 and miR-222 during ischemic conditions (Ribeiro-Rodrigues et al., 2017). The CD34+ stem cells, isolated from mobilized peripheral mononuclear cells from adult human blood, secrete exosomes that can induce angiogenesis in isolated endothelial cells (Sahoo et al., 2011; Haider & Aramini, 2020).

Exosomes deliver proteins and nucleic acids intercellularly without much immunoreactivity making them very suitable as drug carriers. Several organizations like Hamsa Biomed, Exosome Diagnostics, and Caris Life Sciences are developing exosome-based cancer diagnostics (Alvarez-Erviti et al., 2011). Exosomes offer safety, specificity, and stability as a therapeutic delivery system. Exosomes can deliver pharmacologically active compounds to a distantly located specific target site (Tan et al., 2014; Kim et al., 2018b). In general, exosomes are tiny, small vesicles ranging in size between ~30–120 nm. In addition to exosomes, there are other larger extracellular vesicles such as microvesicles (100-1000 nm) and apoptotic bodies (800-5000 nm) that are used to carry biological molecules and dead cells (Todorova et al., 2017). Since there is an overlap in size between these subsets and there is still no consensus on distinct surface markers of each type, the International Society of Extracellular Vesicles (ISEV) use the term extracellular vesicle (EVs) to refer to them collectively (Thery et al., 2018). Due to their tiny size, exosomes easily escape being phagocytosed and reach the area of ischemic injury, making them even more efficient (Kooijmans et al., 2016b). Furthermore, exosomes are relatively stable and less immunogenic due to their autologous nature and small size (Ha et al., 2016; Im et al., 2017). Exosomes do not show tumorigenicity because lack of self-replication and are therefore less toxic and quickly adopted by recipient cells/organs (Stahl & Barbieri, 2002; Laggner et al., 2020; Sun et al., 2020; Gabisonia et al., 2022). Interestingly, exosomes can easily be modified and stored and clear misfolded prion proteins (Cheng et al., 2018; Casado-Diaz et al., 2020). All these characteristics recently make exosomes a promising drug delivery system and a novel therapeutic option in the treatment of various diseases, including cardiovascular diseases.

Although remarkable achievements have been made in this area, research is still growing, and many unsolved mysteries are still to be solved. Before its clinical transformation, several critical limitations need to be addressed. For example, it is necessary to fully uncover the mechanisms regulating exosome loading, packaging, and selection. In addition, it is essential to develop an efficient method for exosome preparation and purification and to modify exosomes for their maximum retention in the body. However, from a clinical perspective, the most challenging obstacle is the targeted delivery of exosomes to the target site/organ via systemic injection. Several elegant reviews have already been published about the function and preparation of exosomes in the cardiovascular system (Kluszczynska et al., 2019; Zhang et al., 2020b; Tiwari et al., 2021). Thus, in subsequent sections, we aim to explore strategies for targeted and therapeutic delivery of exosomes in treating cardiovascular diseases.

2. Exosomes as an alternative therapy

2.1. Tissue-specific targeting

The clinical application of exosome-based therapeutics demands greater exosome specificity in targeting intended tissues. Without this, exosome therapies are at risk of being inefficient and even dangerous. Inefficiency, in this context, refers to the expenditure of resources to engineer therapeutics which do not reach their target organ in large enough volumes to yield a significant difference in patient outcomes. A primary obstacle to ensuring exosome targeting is the rapid clearance of circulating EVs from the body by macrophages, with EVs having a plasma half-life of only 70 to 80 minutes (Chen et al., 2021). Short circulation time reduces the distribution of exosomes within the body, thereby decreasing the chances of their engraftment in target tissues. Moreover, the accumulation of exosomes in unintended tissues may lead to adverse off-target effects. The tissues of most significant concern are those associated with the mononuclear phagocyte system, such as the liver, spleen, lungs, and bone marrow, with exosomes having been found to significantly accumulate in these areas upon intravenous injection (Butreddy et al., 2021). Thus, tissue-specific targeting methods must be developed to ensure the efficiency and reliability of exosome-based therapeutics (Fig. 1).

Fig.1: Schematic illustration of targeting and imaging strategies for exosome-based therapeutic cargo delivery.

Exosome targeting methods involve exogenous and endogenous integration of targeting molecules including free and anchored proteins, nanoparticles, integrins, surface and genetic modifications such as tetraspanins, engineered peptides, receptors and antibodies. These modifications help in the targeted delivery of nanovesicles in the body. The imaging methods involve various labeling strategies to locate exosomal cargo, including bioluminescence, fluorescence, radiotracer, and molecular imprinting. The labeled nanovesicles can be visualized using microscopy, flow cytometry, MRI, and exosome sensing chip.

2.1.1. Direct injection

Given the limitations of circulating exosomes, direct injection of exosomes into the tissue of interest is the most common method used in the past, ensuring the exosomes arrive at their target site. In the heart, the direct injections are primarily performed in either intramyocardial (IM; in the heart wall) or intracoronary (IC; coronary artery) (Zhu et al., 2021). The primary limitation of IM injection is the procedure’s invasiveness, which commonly requires a thoracotomy. Additionally, this method results in an uneven distribution of the therapeutic agent around the injection site. While IC injection is a less invasive, catheter-based approach, it is limited by low retention of the therapeutic agent due to vascular endothelium impermeability and rapid washout. To mitigate rapid washout, a proximal balloon occlusion (BO) technique can be paired with IC injection to slow blood flow near the injection site (Vekstein et al., 2022). Vekstein et al. compared the effectiveness of these three techniques with the delivery of iron oxide nanoparticles in a porcine model. They found IM yielded the most significant retention volume, with 16.0±4.6% of the left ventricle occupied by the nanoparticles, followed by 8.7±2.2% with BO and 0% with IC injection. However, BO was found to allow more excellent distribution of the nanoparticles across the vascular bed; a benefit IM injection could not easily provide (Vekstein et al., 2022).

To improve cardiac retention and procedure invasiveness, Zhu et al. developed an elegant technique in which hydrogels containing exosomes are injected into the pericardium to form a therapeutic cardiac patch. They used a porcine model to deliver MSC-derived exosomes in a porcine heart-derived decellularized extracellular matrix and methacrylic anhydride-hyaluronic acid hydrogels through a minimally invasive procedure consisting of two incisions in the chest used for insertion of an intrapericardial injection needle and a camera. This technique resulted in better exosome retention than intrapericardial injection of exosomes alone (Zhu et al., 2021). Taken together, the above studies highlight different direct injection methods which could be used in the targeted delivery of exosome-based therapeutics to the heart.

2.1.2. Peptide display by genetic modification of parent cells

Despite the advances in direct injection techniques, intravenous injection remains the most common and one of the most convenient routes of administration for exosome (Butreddy et al., 2021). Consequently, technologies must develop to ensure circulating exosomes accumulate at their target site. One such technology involves genetic modification of parent cells to produce exosomes displaying cardiac-specific peptides on their surface. In their study, Kim et al. transfected HEK 293 cells with GNSTM-FLAG-Lamp2b-HA plasmids. The plasmids led to the generation of exosomes expressing CTP-Lamp2b surface protein, with CTP being a cardiac-targeting peptide sequence and Lamp2b being an exosomal membrane protein. CTP-Lamp2 expressing exosomes (CTP-Exo) were isolated and injected into mice tail veins. Compared with control exosomes (CTL-Exo), which represented only Lamp2b, injection of CTP-Lamp2-Exo increased exosome delivery to the heart by 15%, demonstrating the cardiac targeting abilities of CTP-Lamp2b expressing exosomes (Kim et al., 2018a). Another study also showed a LAMP2A dependent loading process of a protein containing KFERQ motif pentapeptide into an exosome’s subpopulation, which was independent of the ESCRT machinery, but dependents on exosome membrane proteins such as CD63, Alix, HSC70, Syntenin-1, and Rab31 (Ferreira et al., 2022).

In a similar study, Mentkowski and Lang transfected cardiosphere-derived cells with pLenti-CMP plasmids to produce Lamp2b-CMP expressing exosomes. CMP is a cardiomyocyte targeting peptide, WLSEAGPVVTVRALRGTGSW, readily entering the target cells. This distinction in cardiac peptides is important as Kim et al. targeted cardiac cells in general, whereas Mentkowski and Lang targeted cardiomyocytes specifically. As such, cardiomyocytes demonstrated increased uptake of CMP-targeted exosomes compared to fibroblasts and HUVECs in vitro. Upon IM injection of exosomes into mice in vivo, CMP-targeted exosomes showed increased retention in the heart after two hours by a 4.3±1.0-fold change compared to control exosomes, which displayed a fold change of 1.9±0.5 compared to PBS control. Thus, this study demonstrated the potential of the Lamp2b-CMP system in the cardiomyocyte targeting (Mentkowski & Lang, 2019). Taken together, these techniques demonstrate the potential to deliver exosomes to the target cells/organs by genetically modifying parent cells to produce modified exosomes displaying cardiac-specific peptides bound to exosomal membrane proteins.

2.1.3. Direct engineering of exosome membranes

Another approach for integrating cardiac-leading peptides or antibodies onto the exosome membrane involves engineering the surface markers onto the existing exosomes. To do this, Antes et al. have developed a three-component anchoring platform and named it “cloaking”. This group has created a synthetic anchoring molecule called DSP cloak using 1,2-bis(dimethylphosphino)ethane (DMPE), polyethylene glycol 5k (PEG), and streptavidin (STVDN). DPS can embed itself within the phospholipid membrane of extracellular vesicles and serve as an anchor for biotinylated molecules. Antes et al. found an efficient homing of DPS anchored extracellular vesicles towards the biotinylated molecules such as antibodies, homing peptides, and fluorophores. Interestingly, the CDC-derived extracellular vesicles (CDC-EV) membranes were embedded with fluorophores along with the antibodies or homing peptides. When neonatal rat ventricular fibroblasts were incubated with CDC-EV cloaked with cardiac fibroblast surface marker (bio-DDR2) and a fluorescent marker (bio-FITC), flow cytometry results revealed a much greater uptake of cloaked bio-DDR2/FITC CDC-EV (30% uptake) compared to control cloaked CDC-EV (5% uptake). They tested the technology with an ischemic targeting peptide sequence and Qdot 655 fluorophores. Following ischemia, cardiac myocytes (CM) were incubated with Qdot 655-cloaked ^IschCDC-EV and found a 13% increase in extracellular vesicles uptake by CM compared to control cloaked CDC-EV. Interestingly, tail vein injection of Qdot 655-cloaked ^IschCDC-EV in the rats following ischemia/reperfusion injury revealed an enhanced uptake of modified EVs in the infarcted regions of the hearts (Antes et al., 2018). Thus, the anchoring technique developed by Andes et al. is a highly versatile technology that could be used to deliver EVs to a defined cardiac cell in various disease conditions.

In another study, a cardiac homing peptide (CHP) was conjugated with dioleoyl phosphatidylethanolamine N-hydroxy succinimide COATSOME^® FE-8181SU5 (DOPE-NHS) to form DOPE-CHP complex. Further, the lipophilic tails of DOPE-CHP are then embedded in the CDC-derived exosome membrane to develop CHP-XOs. Interestingly, conjugated exosomes (CHP-XOs) facilitate its retention to cardiomyocytes compared to control. Better cardiac protection was reported with CHP-XO treatment in the rats following I/R, indicating that the CHP-XOs could be a better strategy to deliver CDC exosomal cargo in the myocardium (Vandergriff et al., 2018). Vandergriff et al.’s study highlights another effective anchoring platform that could be used to engineer cardiac-specific surface markers onto exosome membranes, thereby increasing their heart-targeting capabilities. In addition, several other strategies have been used to insert targeting proteins/ligands in the exosomal membrane for efficient delivery and retention with limited success (Jia et al., 2018). Although these are promising approaches for the targeted delivery of exosomes, for their clinical application, it will be necessary to compare the various anchoring platforms proposed so far to determine their relative stability, safety, efficacy, and costliness.

2.1.4. Cx43 channel-mediated delivery of Exosomal contents

Previous studies have shown that exosomes can deliver their contents to target cells through Connexin 43, the most widely expressed gap junction protein, containing channels in the exosome surface. These channels allow the passage of small molecules such as ions, metabolites, genetic material, and second messengers between adjacent cells (Unger et al., 1997; Sosinsky & Nicholson, 2005; Soares et al., 2015). Connexin 43 is usually expressed by cardiomyocytes and facilitates the propagation of action potentials between cardiomyocytes, although fibroblasts, myofibroblasts, and vascular cells also express it to a lesser extent in the heart (Harris, 2018; Sorgen et al., 2018; Martins-Marques et al., 2019; Rusiecka et al., 2020; Marsh et al., 2021). Irregularities in connexin 43 have been associated with several heart diseases including myocardial infarction (MI) and ischemia-reperfusion (I/R) injuries (Delvaeye et al., 2018; Cocozzelli & White, 2019; Varela-Vazquez et al., 2020). Further, connexin 43 expression is reduced in cardiomyocytes at the infarcted border zone within an hour after ischemic insult (Kieken et al., 2009). Long-term downregulation of connexin 43 also contributes to the increased action potential and thus the potential for ischemic injury (Smith et al., 1991; Peters et al., 1997). Due to the high permeability of connexin 43 channels, it delivers small molecule drugs directly into the cytoplasm of cancer cells, making it an attractive target for therapeutic use (Bonacquisti & Nguyen, 2019). Further, Tania Martins-Marques et al. have demonstrated that the presence of connexin 43 in doxycycline loaded EVs reduce the cardiotoxicity of the drug (Martins-Marques et al., 2016). Therefore, researchers have increased interest in connexin pharmacology and the ability to develop peptides that mimic or bind to connexin 43, known as mimic peptides. These mimetic peptides are cardioprotective against both acute and chronic models of MI and ischemia-reperfusion (IR) injury in rodents (Hawat et al., 2012; Behmenburg et al., 2017; Lucero et al., 2020). Recently, connexin 43-embedded exosome-mimicking lipid bilayers coated chitosan nanoparticles have been constructed which have potential for therapy (Lu et al., 2019). Connexin 43 based synthesis of a more biocompatible and efficient mimetic siRNA delivery platform has opened a new avenue for effective exosome therapy.

2.1.5. Magnetic guiding

One of the limitations that remain with the methods of surface display and cloaking is that they rely on the circulatory system to distribute the therapeutic exosomes to the target organ. Given that the circulatory system transverses the entire body, it will inevitably distribute exosomes far beyond the target organ. A significant chunk of the exosomes may end up at the off-target organs, resulting in a diminished dose efficiency. A solution to minimize this problem involves guiding the exosomes to the region of interest with a magnetic field. Recently Kim et al. packaged iron oxide nanoparticles (IONP) in mesenchymal stem cell (MSC) derived EVs to form magnetic nanovesicles (MNV) and used this strategy to treat ischemic stroke. MNVs have been created by adding IONP to the MSC culture medium, followed by its serial extrusion using membrane filters with increasingly smaller pore sizes. These nanovesicles have similar characteristics with the exosomes including size, cargo loading and drug delivery property but have 100-fold higher production yield. After isolation, MNVs were labeled with a fluorescent dye and injected into mice’s tail vein following I/R injury in the brain’s left hemisphere. Mice wore a magnetic helmet to attract MNV into the brain. IVIS fluorescent imaging revealed increased recruitment of MNVs in the left hemisphere of those mice who wore the magnetic helmet compared to the control (Kim et al., 2020). This demonstrates that applying a magnetic field to attract circulating EVs packed with IONP significantly improves the localization of EVs to the target organs. Although this study was conducted in the context of using nanovesicles to target ischemic stroke injury in the brain, the magnetic guiding technology could be applied to use exosomes to treat cardiovascular diseases in the heart.

2.1.6. Prolonged circulation of exosomes

As described in the preceding section, the primary obstacle for exosome recruitment and retention is its short circulation time. Prolonging the amount of time that exosomes are in circulation increases the likelihood that exosomes administered intravenously will arrive at their target site and is, therefore, an indirect method of promoting the tissue-specific recruitment of exosomes. In this regard, Belhadj et al. have developed a two-phase method they termed “eat me/don’t eat me” to evade clearance of circulating EVs by the mononuclear phagocyte system (MPS) (Belhadj et al., 2020). Phase one employs EVs derived from dendritic cells (DC2.4) with cationized mannan on their surface. DC2.4-derived exosomes were used because they are absorbed by macrophages more readily than exosomes derived from other cell sources; however, cationized mannan helps their better targeting towards the mannose receptor found most on macrophages. The cationized mannan was mixed with DC2.4-derived exosomes to produce extracellular vesicles with cationized mannan on their surface (M-EV). Following tail vein injection, M-EV initiated an “eat me” signal which saturates the mannose receptors on macrophages to preoccupy the MPS during phase two. Phase two employs human serum-derived exosomes expressing membrane protein CD47, which inhibits phagocytosis by macrophages. Therefore, CD47 expressing exosomes release “don’t eat me” signals when injected into the mouse, allowing them to evade rapid clearance from the bloodstream by macrophages. Excitingly, these sheathed exosomes (M-EV+CD47/surface ligand) showed the most significant accumulation in the lung compared to control groups. The sheathing strategy protects tissue-targeting exosomes from rapid clearance in circulation (Belhadj et al., 2020). These results are promising for the development of more efficient exosome doses as well as the reduction of potential off-target effects from the accumulation of therapeutic agents in unintended organs (Belhadj et al., 2020). This “eat me/don’t eat me” strategy can be exploited as a delivery system of treatments to the heart with exosomes.

Along the same line of study, Wan et al. have also developed a two-phase, MPS-suppressing strategy for improving exosome circulation time and better cardiovascular homing by encapsulating exosomes with clathrin heavy chain protein (Wan et al., 2020). In conclusion, direct injection, surface display, membrane engineering, magnetic guiding, and prolonged circulation are all useful techniques that provide significant improvements in the realm of targeted exosome delivery. However, the key to providing the most effective exosome treatments for patients suffering from cardiovascular afflictions will lie in using the above techniques and other techniques in conjunction with each other. Exploiting various techniques to develop increasingly efficient and effective methods of exosomal tissue-specific targeting will be pivotal in applying exosome therapies clinically.

2.2. Exosomes manipulation and sustained delivery system

2.2.1. Hydrogels

Despite being a novel therapeutic model, the biggest hurdle with exosomes is their stability and retention in vivo as the innate immune system rapidly rejects them, and then they accumulate in the liver, spleen, and lungs via the blood circulation (Brennan et al., 2020). To protect exosomes from their failing bioactivity and rapid clearance, hydrogels have been used to immobilize exosomes and release them in a controlled manner (Lv et al., 2019; Xie et al., 2022). Hydrogels are three-dimensional degradable hydrophilic polymer chains with excellent biocompatibility (Hennink & van Nostrum, 2002; Ruvinov & Cohen, 2016; Zhang & Khademhosseini, 2017). Hydrogels can self-assemble into a well-defined structure by employing hydrogen bonds, van der Waals forces, and hydrophobic interactions and are suitable drug delivery systems in an injectable form (Liu & Hsu, 2018). Based on the different sources of polymers, hydrogels are classified into natural, synthetic, hybrid, nanoparticles, and nanotube-based hydrogels (Zhang & Khademhosseini, 2017; Liao et al., 2020; Xie et al., 2022). These different types of injectable hydrogels can promote angiogenesis, reduce cardiac fibrosis and apoptosis, improve scar thickness, and ultimately improve cardiac function.

Natural hydrogels are synthesized using polysaccharides or proteins that can absorb nutrients and small molecules with water with an excellent retention capacity and are generally non-toxic (Ahmed, 2015; Li et al., 2019). Several types of natural hydrogels have been developed, such as extracellular matrix, hyaluronic acid, silk fibroin (sericin), sodium alginate, chitosan, and cellulose hydrogels (Liao et al., 2020). Hyaluronic acid hydrogel has been proven to increase the ejection fraction and the thickness of the left ventricular wall assessed by MRI (Rodell et al., 2016). Another hydrogel, chitosan, has suitable anti-inflammatory property and thus help in the repair of blood vessels in the heart (Dorsey et al., 2015). Sodium alginate hydrogels are non-toxic and easily degradable. Previously they were designed to have a sustained release of angiopoietin, which promotes cardiac repair (Rocca et al., 2016; Hadley & Silva, 2019). Furthermore, silk-sericin hydrogel enhances post-myocardial recovery by promoting inflammation (Song et al., 2016). However, their most significant drawback is that they are expensive and have a long gel formation time. Furthermore, they have an uncontrolled function, less cell adhesion, and rapid degradation (Ahearne, 2014; Pena et al., 2018). Therefore, the focus of research has shifted to synthetic gels.

Unlike natural hydrogels, synthetic hydrogels have many advantages, including robust mechanical properties, desirable function, and less immune rejection (Wang et al., 2017). Synthetic hydrogels can be used to achieve desirable function by adding a new functional group (Pena et al., 2018). For example, crosslinked functional polyion complex regulates nitric oxide release to remove ROS and promote angiogenesis for effective cardiovascular therapy (Vong et al., 2018). A first-in-man pilot study showed that a bioabsorbable stent (IK-5001) was well tolerated without affecting the myocardium post-MI (Frey et al., 2014). Several preclinical efficacy studies have been conducted for treating myocardial infarction using synthetic hydrogels such as Chitosan CSCl-RoY, Type I collagen hydrogel, and TEMPO (Shu et al., 2015; Xia et al., 2015; Zhu et al., 2018). Although synthetic hydrogels have their clinical significance, the lack of cell-specific bioactivities such as cell adhesion, migration, and cell-mediated biodegradation, present its major drawback. To increase site cell adhesion and to maintain cell-to-cell integrity of cardiomyocytes, nanoparticle-based hydrogel fabrication has been introduced. Therefore, searching for clinically suitable injectable hydrogel materials is one of the current research priorities.

2.2.2. Cardiosphere-Derived Cell-Derived Exosomes

Cardiospheres are spherically shaped clusters that can self-assemble and grow on poly D-lysine in semi-suspension culture. In the sphere, cardiac cells grow in a gradient fashion in the peripheral region surrounding the proliferative undifferentiated core cells (Chimenti et al., 2010). A unique feature of cardiosphere-derived cells (CDC) cells is that they can be expanded into manifolds on the surface of the fibronectin monolayer, achieving a suitable cell number for cell therapy. When human cardiac progenitor cells were cultured as CDCs, they were fully capable of in vivo cardiac regeneration and showed improved left ventricular ejection (Chimenti et al., 2010). The use of CDCs post-MI recovery is currently in phase 2 clinical trials and has shown reduced scar size (Chakravarty et al., 2017). Existing data suggest that the therapeutic benefits of CDCs are mainly mediated by secreted exosomes (Ibrahim et al., 2014; Tseliou et al., 2015). Studies have shown that CDC-derived exosomes are sufficient to mediate the full effect of CDCs and not merely act as a component (Tseliou et al., 2015). CDC-derived exosomal miRNAs inhibit both pro-inflammatory and pro-fibrotic pathways upon internalization by macrophages, fibroblasts, and cardiomyocytes (Tseliou et al., 2015). Furthermore, allogeneic CDCs do not induce a significant immune response as they secrete several paracrine factors that reduce the local inflammatory response and infiltration of inflammatory cells into the recipient. Yet, the therapeutic use of CDC-exosomes is advantageous as it recapitulates the entire benefit profile of auto- or allogeneic CDCs without apparent adverse effects (Gallet et al., 2017). It is difficult to predict the actual response to allo-antibodies induced in vehicle-treated and CDC-exosome-treated animals, and what would be the response to repeated treatment with CDC-derived exosomes under such conditions is unclear. Other limitations are associated with the function of CDC-derived exosomes from elderly human patients with cardiovascular risk factors. Their effects may also differ in senescent cells with altered genetic or epigenetic content (Grigorian-Shamagian et al., 2017). L. Grigorian-Shamagian et al. assessed the effects of CDCs on heart structure, function, gene expression, and systemic parameters in aged rats and showed that young CDCs rejuvenate old animals (Grigorian-Shamagian et al., 2017). The life span of the animal used in this study was approximately 2.5 years, and ~22-month-old animals were recruited, so this study may not provide the effects of CDC on longevity. Although this study confirms the role of CDCs and their exosomes in human heart cell longevity in vitro, the major limitation of this model was that it had not included other co-morbidities such as hypertension that likely occur in humans. Second, the gender-specific effects of CDC-exosomes on cardiac cells as a mediator of anti-senescence and their specific antifibrotic and antihypertrophic mechanisms have not yet been explored. Another study showed a better cardiac function in female pigs upon CDC-derived exosome treatment. However, the male gender was not considered in this study. Thus, we cannot make a comparison (Gallet et al., 2017).

Furthermore, CDC-derived exosomes mediated therapy has reversed Duchenne Muscular Dystrophy phenotypes in MDX mice and improved cardiac function (Aminzadeh et al., 2018). Intriguingly, exosomes persisted in this heart for at least 3 months, and a single exosome dose was sufficient to reduce mortality. The therapeutic use of CDC is currently in clinical trials, but the above result encourages CDC-derived exosomes for next-generation therapy (Rogers et al., 2019; Taylor et al., 2019). However, beyond age and gender, CDC-derived exosome therapy has led to improvements in pathologic heart functions in all conditions. Nevertheless, more studies keeping these aspects in mind are needed to understand it better.

2.2.3. Stem/Progenitor Cell-Derived Exosomes

Cardiac progenitor cells (CPCs) have emerged as an effective regenerating therapy in preventing cell damage and improving cardiac repair. CPCs are a promising cell source for cardiac regenerative therapy due to their ability to self-renew in vitro and in vivo and to generate all cardiovascular lineages. (Mol et al., 2017). CPC-derived exosomes have demonstrated therapeutic efficacy in a preclinical ischemic heart model (Orlic et al., 2001). Earlier stem cell-based therapy has been used in cardiac repair. Published data suggest that EVs secreted from various stem cells, i.e., embryonic stem cells (ESCs), human induced pluripotent stem cells (hiPSCs), and hiPSC-derived cardiomyocytes, have shown significant beneficial results in myocardial ischemic models (Khan et al., 2015; Jung et al., 2017). ESC-derived exosomes improved cardiac function post-MI by increasing neovascularization and reducing fibrosis (Ibrahim et al., 2014; Khan et al., 2015). However, some hurdles still need to be overcome for better clinical output. For example, the actual content in stem cell-derived exosomes remains elusive and may pose unknown troubles to recipient cells. Therefore, an in-depth analysis of exosome contents is necessary to ensure the safety of exosome-based cell-free therapy.

2.3. Artificial cargo loading

Given their great potential in treating different cancers, neurological diseases, and cardiovascular diseases, as well as their ability to be successfully loaded with small drug molecules, the demand for exosomes as an ideal drug delivery system is increasing rapidly. The anti-inflammatory potential of curcumin was exacerbated when it was encapsulated in exosomes (Sun et al., 2010). Similarly, researchers have used different strategies to load RNA, siRNA, doxorubicin (anticancer drug), etc., directly into isolated exosomes and tested their clinical benefits (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012; Batrakova & Kim, 2015). Enhanced packaging of miR-125b in BM-MSC-derived exosomes (using miR125b mimic) downregulates inflammatory and apoptotic proteins, resulting in myocardial repair following MI (Chen et al., 2020). Similarly, other studies have shown that MSC-derived exosomes deliver miR-181a and reduce cardiac inflammation post-MI by promoting miRs mediated c-FOS expression and Treg cells polarization (Wei et al., 2019; Gu et al., 2021; Liu et al., 2021; Wang et al., 2021). Both nucleotides and cargo proteins contribute to the remodeling of cardiac function upon myocardial injury. Like nucleotides, the proteins from donor cells can also be genetically engineered or encapsulated directly into exosomes (Kanki et al., 2011; Kooijmans et al., 2016a; Yim et al., 2016). In another study, a tissue matrix metalloproteinase inhibitor 2 (TIMP2) carried by human umbilical cord mesenchymal stem cells-derived exosomes (hucMSC-Exo) significantly improved post-MI cardiac function by activating the Akt/Sfrp2 pathway (Ni et al., 2019). These studies indicate that cargo loading into exosomes through the engineering/modifying donor cells may be a very effective therapy in improving cardiac function post-MI. In addition, several strategies such as electroporation, extrusion, and freeze-thaw cycle have also been developed for cargo loading into exosomes (Fig.2) (Zhang et al., 2020a; Kang et al., 2021; Peng et al., 2021). Furthermore, co-incubation and transfection (transfection reagents) are attractive methods for cargo loading into donor cells (Wang et al., 2018; Song et al., 2019; Wei et al., 2019; Maldonado et al., 2021). In one study, Abreu et al showed that Exo-Fect provided the most promising approach for loading miRNAs to modulate the content of small EVs and, more importantly, it retained their functionality upon delivery into recipient cells (de Abreu et al., 2021). New developments such as engineered chimeric and hybrid exosomes for cargo loading have been developed that may take exosomal therapy to new heights.

Fig.2. Strategies for extracellular vesicles labeling and delivery.

The line diagram shows different cargo loading strategies, including chimeric EVs, direct loading, anchoring, click chemistry, hydrogels, and camouflage and their production. Furthermore, loaded exosomes may provide therapeutic responses to the recipient cells after internalization by fusion, endocytosis, and receptor interactions.

2.4. Labeling and Imaging methods for locating exosomes

It is essential to tag or label the exosomes with specific probes for in vivo or in vitro imaging. Several methods/strategies have been adopted to label exosomes, including covalent binding, genetic modification, membrane integration, encapsulation (electroporation, lipophilic agents, and transporter-dependent), metabolic labeling, and radio labeling (Fig.2). In this section, we will briefly describe some of those strategies.

2.4.1. Covalent binding and Surface modification

A novel chemical approach requires a robust attachment of commercial fluorescent dyes to the surface of a donor vehicle for therapeutic applications. Facilitating the covalent binding of tagging molecules to the exosome surface may provide a better understanding of their pharmacological functions. Recently, a group of researchers successfully visualized the fluorescently labeled exosomes in mice using optical imaging (Gonzalez et al., 2021). They revealed that fluorescent labeling did not alter the original physicochemical properties of the exosomes derived from milk and cancer cells. In this approach, labeled probes containing functional moieties bind covalently with exosomes with minimal dissociation. PEGylation is the most common chemical conjugation in which the exosome surface binds covalently with branched polyethylene glycol (PEG) (Susa et al., 2019). Aminoethyl anisamide-PEG, neuropilin-1, and integrin αvβ3 targeting peptides (c(RGDyK)) are several other examples of exosome surface modification (Chen et al., 2013; Jia et al., 2018; Kim et al., 2018b; Tian et al., 2018b). However, these approaches have several issues, such as labeling may affect the exosomes’ structural and functional properties, leading to unknown binding to recipient cells. For example, exosomes were more distributed in the lungs when tagged with glycosidase. When surface glycosylation was modified by treatment with neuraminidase to remove sialic acid residues, exosomes were optimally accumulated in axillary lymph nodes (Gangadaran et al., 2017b). Therefore, it is necessary to develop a method to analyze the effect of exosome surface modification before using these methods for therapeutic approaches. Genetic modification in the exosomal membrane is another way by which we can overcome the problems related to surface modification.

2.4.2. Genetic modification

Weijia Luo et al. generated a genetic mouse model to track cardiac exosomes using the nano-luciferase-CD63 fusion protein (Luo et al., 2020). This provided a platform for live tracking of tagged exosomes in mice, offering a tool for a range of research applications (Luo et al., 2020). The surface of exosomes can also be modified via exosome-producing genetically engineered cells. These exosomes are more stable with a better yield (Liang et al., 2021). To do so, genes containing target molecules (peptides, receptors, antibodies, etc.) are first combined with the domains of exosomal membrane components (such as tetraspanins, Lamp2b, and the C1C2 domain of lactadherin) and then transfected to the recipient cells (Vakhshiteh et al., 2019). Genetically modified exosome targeting strategies have been applied in various diseases, including cancer and Central Nervous System diseases (Wang et al., 2016). Genetically modified dendritic cells (expressing FasL) derived exosomes reduce inflammation (Kim et al., 2006). Furthermore, conjugation of exosomes derived from cardiac stem cells with cardiac homing peptides via hydrophobic chemicals enhanced the retention of exosomes in the heart (Vandergriff et al., 2018). These results suggest that genetic engineering may help develop hybrid nanocarrier-exosomes that further advance drug delivery systems.

2.4.3. Membrane integration

Labeling exosomal membrane using lipophilic fluorescent dyes is the most widely used labeling method with no toxic effects (Gangadaran et al., 2018). The advantage of this strategy is that lateral transfer of PKH67 lipophilic dye from labeled to unlabeled exosomes is not observed in the co-culture (Nagyova et al., 2014). Therefore, very little background signal noise was observed due to dye transfer to the target cell. But, due to a long in vivo half-life period, target cells may give pseudo signals even after exosome clearance (Gangadaran et al., 2018). For example, PKH2 and PKH26 have around 12 and 100 days of in vivo half-life, respectively (Horan & Slezak, 1989). Exosome aggregation is the major issue with this approach. Another lipid staining dye, Oil red O (ORO), is also very effective in labeling and detecting exosomes (Bharati et al., 2022). This approach is comparatively easy, cost-effective, and gives an intense color to the exosomes. The advantage of this method is that it provides efficient labeling and is very sensitive to real-time tracking of exosome uptake. Further, it does not have long-term persistence, micelle formation, or difficulty in detection.

2.4.4. Encapsulation

Encapsulation is the labeling method that does not require surface modification. Exosomes act as a natural depot to encapsulate biologically active molecules and protect them from their enzymatic degradation (Akbari et al., 2020). In this process, therapeutic agents are either actively or passively incorporated into exosomes (Luan et al., 2017). Several factors assist this encapsulation process, including ceramides, tetraspanins, heat shock proteins, and the endosomal sorting complex required for transport (ESCRT) (van Niel et al., 2018). Other factors such as the miRNA-induced silencing complex (miRISC) and phosphorylated Argonaute 2 (AGO2) help to recognize specific RNA motifs and facilitate their encapsulation (van Niel et al., 2018). The encapsulation process is mainly mediated by electroporation, lipophilic agents, or via transfer-dependent mechanisms (Gangadaran et al., 2018). This labeling method has some associated limitations, such as electroporation can cause aggregation while lipophilic and transfer-dependent encapsulation can give a background signal. The lipophilic encapsulation process may potentially sustain the release of encapsulated probes from exosomes. Transfer-dependent encapsulation requires a transporter (GLUT1) protein to carry the therapeutic agent. However, uneven distribution of transporter proteins on different exosomes may cause uneven loading or encapsulation of the agent, causing background signals from the released probes (Jung et al., 2018). The particular cell type may also limit the expression of a specific transporter protein.

2.4.5. Metabolic labeling

Incorporating metabolic chemistry into exosomes brings remarkable innovation to their mechanism of action, making it a very advanced tool for biomedical studies. Metabolic labeling can be achieved by adding specific substances during the cell culture process. After isolation of the metabolically labeled exosomes, covalent binding of the probe is performed (Wang et al., 2015; Lee et al., 2018). However, this external modification during cell culture may affect the specific properties of exosomes during the ultrafiltration process. Lee et al. developed a new facile exosome labeling by incorporating azide-containing sugars in glycan glycoprotein via a strain-promoted azide-alkyne click reaction (SPAAC) (Lee et al., 2018). In this process, Tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) is incorporated into glycans spontaneously within the cell when cultured, which is then distributed onto exosomes. In addition, the fluorescent dye was tagged in the azido-containing exosomes (Lee et al., 2018). Lin Zhu et al. have shown that metabolic labeling holds excellent potential for real-time imaging of exosomal protein-specific glycosylation and for studying the role of exoPD-L1 glycosylation in immunosuppression.

2.4.6. Radiolabeling

The exosome labeling methods discussed above require fluorescence- or luminescence-based imaging systems for their in-situ tracking. However, two major drawbacks are the poor ability to acquire images in deep tissues and signal weakening over time (Gangadaran et al., 2017a). Recently advanced labeling and identification technologies, including positron emission tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI), allow exosomes to be tracked deep in the tissue as well as monitoring the biodistribution and pharmacokinetics of exosomes (Wen et al., 2016; Choi et al., 2022). Although radioactive isotope labeling was better than the radiolabeled approach because it was more durable and reliable for in vivo studies (Bindslev et al., 2006; Wang et al., 2014). Additionally, radiolabeled imaging systems with excellent differentiation capability are potentially harmful to targeting exosomes and host tissues. Therefore, these strategies cannot be suitable for in vivo studies.

Surface radiolabeling is most common among other exosome radiolabeling. Several strategies incorporate radionucleotides into the exosome membrane, including nucleotide integration directly into the membrane or into a membranous protein, genetic modifications, or attachment via chelator (Khan & R, 2021). Chemical modification of exosomal membranes or membranous proteins is the major limitation of surface radiolabeling that impairs the surface integrity of exosomes (Clayton et al., 2003; Clayton et al., 2004; Liu et al., 2012; Hu et al., 2020; Skotland et al., 2020).

Intraluminal radiolabeling is another strategy for radiolabeling in which the radiotracer is completely entrapped in the intra-vesicular space of the exosome. And thus, like surface radiolabeling, it does not involve trans-chelation of radionucleotide and avoids these effects from serum proteins. Delivery of the radiotracer into the exosome lumen is carried out by remote loading or ionophore-chelator binding. In remote loading, the lipophilic radiotracer first enters the lumen and is converted to be hydrophilic in nature by a specific endogenous intravascular glutathione and finally remains in the exosomal aqueous core (Phillips et al., 1992). Ionophore-chelator binding is another delivery system that utilizes ionophore ligands. Tropolone and 8-hydroxyquinoline (auxin) are the best examples of ionophore ligands that complex with radiometals and facilitate their transport through lipid membranes into the exosome lumen (Edmonds et al., 2016). The potential disadvantage of this approach is that it is difficult to ascertain to which exosomal material this radiolabeled nucleotide will bind, further complicating in vivo image interpretations (Man et al., 2019; Khan & R, 2021).

2.4.7. Immunogold-labeling method

Immuno-gold labeling is an indirect immunofluorescence staining technique mainly used to visualize exosomal membranes using TEM or cryo-TEM. In this process, specific cellular components of interest within exosomes are labeled by gold-conjugated antibody proteins. Immunogold reagents are built around colloidal gold particles and viewed directly under an electron microscope. The distribution of glucose transporter proteins (GLUT-1 and GLUT-4) has been successfully studied in cardiac tissue following stimulation by insulin or ischemia using the immunogold labeling (Davey et al., 2007). Recently, gold nanoparticles (Au NPs) in exosomes were used to detect Frizzled-10 protein in gastrointestinal cancer for its early diagnosis (Scavo et al., 2020). To see the presence of proteins acting as cancer-associated markers with primary antibodies to the FZD10 protein, a TEM grid was used to support depositing Au NPs on the surface of the exosomes (Lane et al., 2018). However, this method does not give quantitative results; non-target molecules also give false results (Davidson et al., 2022; Su et al., 2022).

3. Limitations and challenges

While exosomes are a promising vehicle for treating cardiovascular diseases, hurdles toward clinical application of exosome therapy remain. The type of exosome therapy often discussed is stem cell-derived exosomes (Adamiak & Sahoo, 2018). The most appealing aspect of stem cell-derived exosomes is their innate therapeutic capabilities, similar to those of their parent cells. In this regard, these exosomes are effective and convenient for treating various conditions. However, the challenge that soon arises is that natural exosomes carry a plethora of biomolecules with unknown effects (Li et al., 2021). For example, miRNA, a common exosome cargo, allows a single miRNA to regulate multiple genes, possibly having numerous off-target effects (Kilikevicius et al., 2022). Additionally, cells release exosomes with varied cargo depending on changes in environmental conditions, thereby further complicating the predictability of natural exosome therapies. Therefore, quality control protocols are necessary to safely prescribe stem-cell-derived natural exosome treatments to ensure homogeneity among exosomes. This permeates every stage of the production process, including cell cultures, exosome isolation, characterization, modification, and storage (Adamiak & Sahoo, 2018). In different diseases, exosomes have been isolated from various body fluids, including saliva, semen, blood plasma, bile, gastric juice, and breast milk (Caby et al., 2005; Ogawa et al., 2008; Lasser et al., 2011; Li et al., 2016; Choi et al., 2017; Madison et al., 2017). Since exosomal yield significantly varies across age, sex, disease severity, and individual lifestyle, a separate optimized protocol is necessary for each type of specimen. Furthermore, the different degradation status of these samples makes it crucial to know how to save exosomes during the isolation process (Pritchard et al., 2012). Several methods are used to isolate exosomes, such as ultracentrifugation, immunoaffinity, density gradient, microfluidics, size exclusion chromatography, and many others (Caby et al., 2005; Raposo & Stoorvogel, 2013; Liga et al., 2015; Nordin et al., 2015; Gardiner et al., 2016). However, these methods have their advantages and disadvantages. Careful selection of the appropriate isolation method for the particular sample will be critical. In addition, exosome storage is crucial because it degrades during freeze-thaw conditions and can lose its contents. The preservatives such as EDTA, heparin, or citrate carried with them during isolation also affect their stability significantly (Wisgrill et al., 2016). After careful isolation and storage, the next hurdle is to characterize the exosomes, which is a critical step in knowing their dosage for therapeutic use (Gilani et al., 2017; Pedersen et al., 2017). At this step, the researcher adopted various methods, including the beads selection method, polycarbonate membranes, CD63 expression using ELISA, and flow cytometry for their size exclusion which caused considerable variation in the result (Gilani et al., 2017; Pedersen et al., 2017). Researchers have also used several other techniques to characterize exosomes based on size and number, such as electron microscopy, nanoparticle tracking analysis, and flow cytometry. However, these methods provide inconsistent results. Thus, exosome characterization strategies need to be standardized across general practice. Additionally, all these protocols must be scalable to make possible the production of exosomes in a clinically relevant abundance. Given the coordination of efforts and monetary input such a sophisticated system requires, exosome-based therapies face an upward climb toward widespread implementation. Moreover, despite advances in the tissue targeting capabilities of exosomes, exosomes continue to accumulate in the liver and spleen and have relatively short plasma half-lives. This poses problems regarding dose efficiency and off-target effects but seems a manageable obstacle if various tissue-targeting strategies are used in conjunction. Along with this, exosome tracking in vivo may lead to a clearer understanding of exosome biodistribution and uptake, thereby facilitating advancements in tissue targeting strategies (Adamiak & Sahoo, 2018).

4. Conclusions

Despite the great potential of exosomes in biomedical applications, the limitations discussed above discourage their use in biomedical therapy so far. Researchers are using live imaging of exosomes to study the biological phenomenon in various diseases such as cancer, neuronal, and heart disease. Since different types of reporters are used for exosome labeling and imaging, an in-depth study of each reporter is necessary. Simultaneously, it is necessary to reduce the rate of false positive reporter signals and characterize the actual spatiotemporal property of labeled exosomes. There is currently no definitive method to identify exosome subpopulations carrying different information secreted by the same cell line under physiological and pathological conditions. Given the recent innovations in exosome research, we are sure that in the coming days, a thorough understanding of the exosome’s in vivo pharmacokinetics and biodistribution will be possible. Furthermore, technological advances exploring exosome characterization, specific drug loading and imaging in a safe manner, their targeting to the heart, and real-world implications of exosomes will facilitate the use of exosomes to treat cardiovascular diseases.

Biographies

Suresh Verma Ph.D. is an Associate Professor in the Division of Cardiovascular Disease, Department of Medicine at the University of Alabama at Birmingham, Alabama, USA. Before joining UAB, Dr. Verma served as an Assistant Professor in the Feinberg Cardiovascular Research Institute, Northwestern University Chicago, and later at the Center for Translational Medicine, Temple University Philadelphia. The primary mission of Dr. Verma’s research is to identify and test novel therapeutic targets to prevent or delay the development and progress of heart failure.

Prabhat Ranjan Ph.D. is a postdoctoral fellow in Dr. Verma’s laboratory. He is the recipient of the prestigious AHA postdoctoral fellowship. His research aims to understand exosome-mediated crosstalk within cardiac cells.