Application of exosomes in the treatment of cardiovascular diseases

Application of exosomes in the treatment of cardiovascular diseases.  EVs carry molecules such as proteins, RNA and/or microRNAs (miRNAs), which act as carriers in cell-to-cell communication.

Preface

For decades, despite the discovery of many new breakthrough therapies, cardiovascular disease is still the world’s leading cause of morbidity and mortality. In the past 20 years, some therapeutic interventions have been clinically applied, including cell-based therapy; however, the survival rate and implantation rate of cell transplantation in the ischemic environment of heart tissue are low, which limits their clinical efficacy . From a mechanistic point of view, the reasons for the functional improvements observed in cell therapy are not yet clear; however, experimental data suggests that they may act through paracrine, mediated by exosomes (EVs) or other factors. Therefore, there is an increasing interest in the development of cell-free therapies based on exosomes.

EVs are extracellular structures surrounded by lipid bilayers and are secreted by almost all known cell types. EVs include two major types of exosomes and microvesicles. Extracellular bodies (30-150nm in diameter) are intraluminal vesicles formed by the membrane invagination of polycystic endosomes, which are released into the extracellular space after the polycystic endosomes fuse with the cell membrane. Microbubbles (50-1000nm in diameter) are a group of highly heterogeneous EVs, which are characterized by sprouting outward through the plasma membrane to produce and secrete.

EVs carry molecules such as proteins, RNA and/or microRNAs (miRNAs), which act as carriers in cell-to-cell communication. A large amount of evidence indicates that EVs are involved in many physiological and pathological cardiovascular processes, including the regulation of angiogenesis; blood pressure control; cardiomyocyte hypertrophy and/or survival; and myocardial fibrosis. Since EVs are commonly found in body fluids, such as blood and urine, EVs have been used as potential biomarkers for cardiovascular disease. In addition, because EVs are an important part of the paracrine effect of stem cell therapy, EVs can be used as an independent therapy for cardiovascular diseases. Preclinical studies have shown the therapeutic potential of EVs in protecting the heart from ischemic damage and promoting angiogenesis.

Biophysical properties of EVs

Size:

Size is one of the parameters used to classify EVs. Generally speaking, extracellular bodies are smaller than microbubbles and have a more uniform size distribution. The size of EVs is very important for its research and application, because most of the separation and identification steps depend on the density and diameter of EVs. In addition, the size of EVs is determined by their way of occurrence and is related to the components of EVs. Finally, the tissue biodistribution, cell internalization, and intracellular transport efficiency of EVs depend on size. For example, after administration, large (>200nm) and aggregated exosomes will stay in the lung, liver, and spleen, and eventually be engulfed by macrophages or interact with non-vascular cells and tissues due to impermeability.

At the cellular level, particles of different sizes can induce different uptake mechanisms. For example, particles with a diameter of 100 nm can be absorbed by clathrin-mediated or cytoplasmic membrane microcapsule-mediated endocytosis, while larger aggregates are more likely to be directed to lysosomal degradation or membrane recycling. Therefore, smaller vesicles may be more efficiently delivered into the cell. In the cardiac environment, especially for systemically administered EVs, this factor is critical because EVs must successfully penetrate into the heart tissue and then be effectively absorbed by the relevant cell types.

Charge:

Another important characteristic of EVs is surface charge. Because part of the EV membrane comes from the phosphate-rich plasma membrane (the rest comes from other organelles, including the Golgi apparatus), EVs usually have a net negative charge, as do cells. However, the charge is also highly dependent on the sugar composition of the plasma membrane, which in turn depends on the expression level of sialyltransferase (enzyme that converts sialic acid into oligosaccharides) in the endoplasmic reticulum and Golgi. The change in surface charge can be used to infer the stability of EVs in a suspended state, because a lower absolute value may reduce the repulsive force and make EVs more likely to aggregate. The size and surface charge of EVs are crucial for determining the interaction mechanism between EVs and many potential ligands and their absorption by target cells. Finally, the presence of contaminants in EV samples (such as protein or lipid aggregates) may affect various functions and parameters. Taking into account the uneven surface charge of these pollutants, aggregates will form between them and EVs. Therefore, a suitable purification protocol must be followed.

Membrane composition:

The cholesterol content of extracellular body membranes is higher than that of donor cell membranes, which makes them less susceptible to penetration by small molecules. In addition, compared with the donor cell membrane, the extracellular membrane contains higher levels of phosphatidylserine, glycosphingolipid and sphingomyelin, while the content of phosphatidylcholine is lower. Compared with microvesicles, extracellular bodies have a lower protein-to-lipid ratio. In fact, high cholesterol and sphingolipid content makes extracellular bodies more resistant to detergents and high temperatures than microbubbles.

In addition to lipid components, extracellular bodies are also modified by proteins and sugars. These proteins and sugars help maintain the charge and membrane structure of the exosomes, and mediate the interaction between the exosomes and target cells. For example, tetraspanins are a class of membrane proteins that are abundant in extracellular bodies, some of which (CD9, CD63, and CD81) are considered to be general markers of extracellular bodies. Functionally, tetraspanins are involved in membrane fusion and cell adhesion, and therefore play an important role in extracellular internalization.

Other types of proteins, such as chemokine receptors (e.g., CXC chemokine receptor 4), adhesion molecules, and proteoglycans (e.g. heparan sulfate proteoglycan), have been shown to mediate EV and cell surface Play a role in the interaction. When these protein and sugar-based components are removed or masked, EV internalization and biodistribution will change. The transmembrane protein of EVs has the same structure as the secretory cell, which gives a certain degree of cell characteristics and possible chemotaxis.

Inclusions:

Since the end of the 1990s, EVs have been considered important mediators of cell-to-cell communication, especially in terms of immune response and cancer. In 2007, it was discovered that exosomes contain miRNAs and other types of RNA, and can transfer their contents to target cells, ultimately affecting the activity of these cells. This concept was further verified in 2007. High-resolution density gradient fractionation and direct immunoaffinity analysis were used to further analyze the components of EVs. EVs contain a variety of proteins and RNA in the lumen, including long and small non-coding RNA, transfer RNA and ribosomal RNA. Extracellular bodies do not contain DNA, although DNA may be present in larger EVs or samples rich in extracellular bodies due to co-precipitation with histones. Proteins can enter EVs through post-translational modifications (such as ubiquitination and glycosylation), and these pathways can be used to carry targeted proteins into EVs. Research on the functionally established interaction network between EV proteins shows that the proteins in the vesicles form a “nano universe” in a highly ordered manner, rather than just a disordered collection of “intracellular fragments”.

Natural EVs for the treatment of cardiovascular diseases

The first study using EVs as a potential therapeutic intervention for cardiovascular disease was published in 2010. At the beginning of the 21st century, some research groups showed that transplanting different types of cells, including mesenchymal stem cells and hematopoietic progenitor cells or stem cells (CD34+ cells), can improve heart repair after myocardial infarction (MI). Subsequently, the positive effects of these stem or progenitor cells proved to be mediated not by direct contributions from implanted cells, but by paracrine factors, especially EVs secreted by surviving cells.

Since these pioneering studies, several research groups have demonstrated the regenerative properties of EVs secreted by stem or progenitor cells and differentiated somatic cells in the case of MI, limb ischemia, and chronic skin injury. Some studies have been conducted in atherosclerotic sclerosis and stroke, but most of the cardiovascular studies on EVs are related to ischemic heart disease and MI. EV tracking studies have shown that, compared with intracoronary or intravenous administration, intramyocardial administration of EVs can produce higher EV retention in the heart. The preclinical data collected so far show that, regardless of its source, EVs can improve the left ventricular ejection fraction (1.3 times greater than the untreated group) and reduce the area of ​​myocardial infarction (reduced by 3 times compared with the untreated group). The therapeutic effects of EVs and donor cells have been evaluated, and the results collected so far show that in the case of MI, EVs are as effective as donor cells.

The therapeutic effects of EVs in recipient cells are mainly due to the delivery of proteins and/or non-coding RNAs, especially miRNAs. For example, miRNAs considered to be involved in the cardiovascular protection of extracellular bodies include miRNA-19a-3p, miRNA-21, miRNA-22, miRNA-24, miRNA-29a, miRNA-126, miRNA-143, miRNA-146, miRNA-181b, miRNA-210, miRNA-222 and miRNA-294-3p, etc. In previous cell therapy studies, some miRNAs (including miRNA-19, miRNA-21, miRNA-24, and miRNA-210) have been confirmed to be related to cardiovascular repair, while other miRNAs are newly discovered.

Another important component related to the biological activity of EVs is protein, such as platelet-derived growth factor D and pappalysin1. Some of these proteins are located on the surface of the EV and therefore do not need to be delivered to the cytoplasm of the recipient cell. For example, pappalysin 1 is highly expressed in the extracellular body of cardiac progenitor cells. Pappalysin 1 cleaves insulin-like growth factor binding protein 4 into insulin-like growth factor 1 (IGF-1), which then activates IGF-1 receptors, leading to AKT , ERK1 and ERK2 phosphorylation and subsequent caspase activation and reduction of cardiomyocyte apoptosis, thereby mediating the cardioprotective and angiogenic effects of EVs derived from cardiac progenitor cells.

According to the source and content of EVs, they can trigger various cardioprotective effects, such as improving the survival of cardiomyocytes and endothelial cells by regulating autophagy; activating pro-survival signaling pathways (such as signaling pathways involving AKT, ERK and Toll-like receptors) and Decrease of oxidative stress level; regulate inflammatory response by influencing immune cell polarization (ie induce a more restorative state rather than an inflammatory state) and cytokine secretion and increase the activation of CD4+ T cells; reduce scar content; angiogenesis stimulate. For example, heart-derived EVs improved the cardiac function of MI mouse models through miRNA-146, reduced cell apoptosis and inflammation, and increased cardiomyocyte proliferation and angiogenesis. The extracellular matrix-derived EV carries miRNA-199a-3p, which restores the electrical function of the bioengineered atrium by regulating the acetylation of the transcription factor GATA4.

Bioengineering of EVs

The above studies emphasize the potential of natural EVs in cardiovascular therapy. However, their clinical potential has not been recognized, and important limitations must be overcome before they can become an effective treatment tool. These limitations can be solved by using bioengineering techniques to improve the performance of EVs. Therefore, in order to improve the efficacy of natural EVs in the treatment of cardiovascular diseases, people have developed technologies to regulate EVs and improve their biological activity, stability, targeting and delivery to the cardiovascular system (through the development of EVs delivery systems).

Tracking:

Tracking EVs in vivo and tracking their biodistribution is of great significance for evaluating their cardiovascular therapeutic potential. Fluorescence, luminescence, PET-MRI and SPECT imaging techniques have been used to monitor EVs in vivo, usually after separating EVs with chemical ligands. Although fluorescence and luminescence imaging techniques are easy to operate, they cannot provide high sensitivity or absolute quantification. In contrast, methods that rely on PET-MRI or SPECT-CT provide higher sensitivity and absolute quantification, and also allow the acquisition of images with anatomical details. In general, intravenous injection of labeled EVs isolated from different cell sources (without further modification other than the labeling) will not cause damage to the heart. However, the accumulation of EVs in the heart is affected by the delivery route, delivery concentration and the characteristics of EV secreting cells.

Biological activity:

① Regulate EV secreting cells. EV-secreting cells can be regulated by two different procedures: culture and transfection of exogenous compounds (such as nucleic acids, especially miRNAS23, miRNA antagonists, Y RNA) under stress-induced conditions (such as hypoxia, serum starvation or inflammation) , Plasmid DNA and small molecules to increase its biological activity).

For example, EVs collected from rat cardiac progenitor cells cultured under hypoxic conditions increased the ability of cardiac endothelial cells cultured in vitro to form tubular structures and reduced the amount of cardiac fibroblasts cultured in vitro compared with normally collected EVs. T

he expression of pro-fibrosis genes improves the function of the infarcted heart. In addition, EVs collected from human bone marrow mesenchymal stem cells transfected with miRNA-181a increased the repair status of peripheral blood mononuclear cells, and were injected into the heart of mice suffering from ischemia-reperfusion injury in the myocardium. In comparison, EVs rich in miRNA-181a increased the left ventricular ejection fraction by 12%.

② EV secreting cells can also be adjusted by changing the medium. For example, the level of miRNA-31 in EVs collected from human adipose stem cells cultured in endothelial differentiation medium increased. Compared with EVs collected from adipose-derived stem cells grown in normal culture medium, the resulting EVs promoted endothelial cell migration, lumen formation and aortic ring growth. Some cell platforms have been developed to customize the enrichment of EVs with specific proteins and RNA; however, these platforms have not been evaluated for cardiovascular disease applications. In addition, few studies have used cellular mechanisms to design ev with specific epitopes to target the heart.

③ Modification method after separation. The advantage of using cell modification mechanisms to produce EVs is that it retains most of the biophysical properties of EVs, but it also has disadvantages-overexpression of a specific molecule in the cell may have unpredictable consequences for its biology and eventually interfere with the development of EVs. Biogenesis. The strategy of using post-isolation modification of EVs provides an alternative method for effective bioengineering loading, targeting and delivery of EVs, regardless of the cells from which they originate. However, the method of post-isolation modification may obscure or impair the endogenous EV characteristics and ultimately impair the biological activity of the EV.

Membrane permeation strategies, such as electroporation, heat shock or freeze-thaw procedures, detergent treatment, and ultrasound, to load foreign materials, have been applied to EVs, with uneven success rates. Other strategies include passive loading, using the hydrophobicity of EV membranes to passively load compounds of interest into EVs, and modifying molecules of interest with cholesterol. These studies report a load efficiency of up to 70%, but it has a negative impact on the production of EVs.   The influence of physical properties and the exact mechanism of action remain to be elucidated.

Several EV preparations were enriched using transfection agents or membrane permeability strategies and evaluated in the context of cardiovascular disease to reduce cardiac fibrosis, regulate inflammation, and increase angiogenesis. For example, miRNA-21-5p plays a crucial role in the development of fibrosis after myocardial infarction. It regulates multiple gene targets including MMP2, PTEN, and SMAD7. Compared with the use of unmodified EVs, human peripheral blood-derived EVs rich in miRNA-21 inhibitors can reduce fibrosis in MI mouse models. In a separate study, EVs were collected from cardiac progenitor cell-derived cells, enriched in miRNA-322 by electroporation, and systematically injected into MI mouse models. Compared with unmodified EVs, it reduced Myocardial infarction size and fibrosis, and increase angiogenesis. In summary, these studies have proved the possibility of enriching EVs after separation, thereby improving their biological activity compared with unmodified EVs.

Distribution and biological targeting:

After systemic administration in animal models, EVs are quickly cleared or concentrated in the liver, spleen and lungs. The half-life of EVs (usually in the range of minutes) essentially depends on the source of EVs secretory cells . The biodistribution of EV is affected by many factors, including the route of administration and dosage. As previously observed in cell therapy, EVs retained in multiple organs may induce systemic anti-inflammatory effects and improve the regeneration capacity of the cardiovascular system. However, research clearly shows that the improvement of EV efficacy is related to the retention of EV in the diseased area. Therefore, several strategies have been developed to control the biodistribution of EVs and target EVs to specific organs and tissues.

One strategy to maximize the uptake of EVs is to increase their stability in the circulation, thereby increasing the possibility of EVs interacting with target cells or tissues. Modification of EVs with polyethylene glycol increases their circulation time and reduces their uptake by non-specific cells. Another strategy relies on the modification of EV membranes with specific proteins or peptides that can interact with specific cell receptors or extracellular matrix components expressed in the cardiovascular system. Some examples include cyclic peptides (RGDyK) targeting cerebral ischemia, lysosomal-associated membrane glycoprotein 2B (LAMP2B)-RVG structures targeting neurons, CSTSMLKAC peptide targeting ischemic myocardium, and cardiomyocytes targeting The WLSEAGPVVTVRALRGTGSW peptide. Tetraspanins proteins exist in EV membranes and preferentially bind to specific cell lines (for example, Tetraspanins proteins bind to integrin α4β4 chains expressed by endothelial cells), but these proteins may not be sufficient to selectively and effectively target EVs to organs or tissues.

In heart disease cases, intramyocardial administration of EVs has been reported in preclinical studies; however, this route of administration is not always clinically advisable because it involves catheterization. Intravenous injection of EVs is a much simpler procedure and allows repeated applications; however, this method is more likely to deviate from target binding and increase the possibility of adverse reactions. The modified EVs may eventually overcome these obstacles and deliver their therapeutic drugs to the injured heart. There are two methods used to modify the surface of EVs to target the heart. In one approach, EV-secreting cells are genetically modified to express peptides, which are then incorporated into the secreted EVs membrane. For example, EV secreting cells have been genetically modified with a lentiviral vector that expresses a membrane protein (LAMP2B) fused with a peptide (CSTSMLKAC) to target ischemic cardiomyocytes. Although there is no absolute quantification of EVs accumulation in the heart, fluorescence imaging shows that peptide-modified EVs accumulate more than EVs without surface modification.

In another method, the surface of EVs can be chemically modified by two main strategies: physical binding of lipids modified with proteins (such as streptavidin) or peptides into the membranes of EVs, and chemical modification of the linker The functional group (carboxyl or amine group) coupled to the surface of EVs. These reactions can be carried out in aqueous solutions, which are fast, selective and very efficient compared with traditional bio-combination techniques. For example, injecting EVs modified with myocardial targeting peptide (CSTSMLKAC) into mice after myocardial infarction increased the left ventricular ejection fraction by 46%.

The genetic modification and chemical modification of EVs secreting cells have their own advantages and disadvantages for EVs. Genetic methods may produce a more standardized product, which is desirable to meet regulatory expectations. However, this strategy has some limitations, including changes in the biological activity of EVs due to genetic manipulation, and difficulty in controlling the density of targeted epitopes and their glycosylation status. Chemical methods can allow effective control of the structure of EV surface modification (including unnatural amino acids that prevent peptide degradation) and the density of targeted epitopes, regardless of the cell from which it originated. In addition, chemical methods can be carried out during the purification process of EVs, so it is more suitable for clinical applications.

Uptake and intracellular transport:

The use of fluorescent imaging technology and labeled EVs can be used to study the internalization and intracellular transport of EVs. The endocytosis of EVs seems to be affected by the interaction of EVs with the cell membrane and the endocytosis capacity of the recipient cells. The internalization of EVs can be mediated through non-specific interactions, or through specific interactions, such as receptor-dependent pathways. Little is known about the difference in the endocytosis capacity of cells, the effect of EV surface modification on intracellular transport, and the effect of EV surface modification that may increase endolysosome escape. However, these issues are critical because most internalized EVs are processed in the endolysosome pathway and eventually degraded in the lysosome. In fact, approximately 60% of internalized EVs localize to lysosomes after 48 hours of contact with recipient cells.

Someone proposed a strategy to increase the escape of lysosomes in EVs. In one approach, EVs are coated with cationic lipids and pH-sensitive fusion peptides, which increase the destruction of the endolysosome membrane, resulting in effective cytoplasmic release of EV contents. In another method, EVs are encapsulated by arginine cell-permeable peptides to induce active micropinocytosis and release EVs into the cytoplasm more effectively.

Delivery:

Local administration of EVs at the injury site can increase the possibility of targeted cells and target cell uptake. Several strategies based on biomaterials have been developed to continuously release EVs at the injury site, including hydrogels containing hyaluronic acid, alginate, chitosan, collagen, or amphiphilic peptides. The design of the hydrogel composition takes into account its biology, degradation, in-situ gelation characteristics, mechanical and EV release characteristics. There are several ways to incorporate EVs into the hydrogel. In one approach, the EV is mixed with the polymer solution without involving a reaction between the two entities. Then the solution is injected into the tissue of interest, and within a few minutes, the polymer is physically cross-linked through intermolecular interactions, intermolecular ionic interactions, or a self-assembly process mediated by polymer hydrophobic chains, thereby keeping EVs in Within the polymer structure.

In another method, EV is mixed with a polymer solution to form a polymer-EV conjugate and initiate the chemical cross-linking process. The solution is then injected into the tissue of interest to further crosslink and form a hydrogel. In the third method, EVs are physically incorporated into the hydrogel after polymerization. Compared with EVs without a sustained-release system, the EV release hydrogel increases the left ventricular ejection fraction, reduces the infarct size, and reduces the burden of arrhythmia.

For example, the left ventricular ejection fraction of the infarcted heart of rats treated with the hydrogel patch containing EVs was 40% and 25% higher than that of the control group without EVs and the control group that received EVs alone. Importantly, the kinetics of EV release from the hydrogel seems to play an important role in its therapeutic effect. For example, the slow release of EVs by hydrogels implanted in mouse skin wounds is not as effective as the use of remote trigger hydrogels to synergistically release EVs during skin regeneration. In this study, EVs were immobilized in a hydrogel that can be lysed by exposure to blue light. Then the wound is exposed to blue light at regular intervals (this can be changed according to the healing rate of the wound) to trigger partial hydrogel degradation and controlled release of EVs.

Outlook

In the past ten years, significant progress has been made in understanding the biological characteristics of EVs and their applications in the cardiovascular field. Targeted technology can increase the accumulation of EVs in the cardiovascular system, thereby reducing the required dose.

The strategy of using specific biomolecules to increase the content of EVs may be the key to its successful clinical application. The use of EVs tracking technology will improve our understanding of the biodistribution mechanism of EVs.

In addition, loading foreign molecules on EVs and controlling their release in vivo will create opportunities to improve the biological activity of EVs. Finally, bioengineered EVs will be a promising, cell-independent, durable and customizable treatment to improve the outcome of cardiovascular disease patients.

(source:internet, reference only)

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