Exosomes for angiogenesis induction in ischemic disorders

Abstract Ischaemic disorders are leading causes of morbidity and mortality worldwide. While the current therapeutic approaches have improved life expectancy and quality of life, they are unable to “cure” ischemic diseases and instate regeneration of damaged tissues. Exosomes are a class of extracellular vesicles with an average size of 100–150 nm, secreted by many cell types and considered a potent factor of cells for paracrine effects. Since exosomes contain multiple bioactive components such as growth factors, molecular intermediates of different intracellular pathways, microRNAs and nucleic acids, they are considered as cell‐free therapeutics. Besides, exosomes do not rise cell therapy concerns such as teratoma formation, alloreactivity and thrombotic events. In addition, exosomes are stored and utilized more convenient. Interestingly, exosomes could be an ideal complementary therapeutic tool for ischemic disorders. In this review, we discussed therapeutic functions of exosomes in ischemic disorders including angiogenesis induction through various mechanisms with specific attention to vascular endothelial growth factor pathway. Furthermore, different delivery routes of exosomes and different modification strategies including cell preconditioning, gene modification and bioconjugation, were highlighted. Finally, pre‐clinical and clinical investigations in which exosomes were used were discussed.


Abstract
Ischaemic disorders are leading causes of morbidity and mortality worldwide. While the current therapeutic approaches have improved life expectancy and quality of life, they are unable to "cure" ischemic diseases and instate regeneration of damaged tissues. Exosomes are a class of extracellular vesicles with an average size of 100-150 nm, secreted by many cell types and considered a potent factor of cells for paracrine effects. Since exosomes contain multiple bioactive components such as growth factors, molecular intermediates of different intracellular pathways, microRNAs and nucleic acids, they are considered as cell-free therapeutics. Besides, exosomes do not rise cell therapy concerns such as teratoma formation, alloreactivity and thrombotic events. In addition, exosomes are stored and utilized more convenient. Interestingly, exosomes could be an ideal complementary therapeutic tool for ischemic disorders.
In this review, we discussed therapeutic functions of exosomes in ischemic disorders including angiogenesis induction through various mechanisms with specific attention to vascular endothelial growth factor pathway. Furthermore, different delivery routes of exosomes and different modification strategies including cell preconditioning, gene modification and bioconjugation, were highlighted. Finally, pre-clinical and clinical investigations in which exosomes were used were discussed.

K E Y W O R D S
angiogenesis, exosomes, hypoxia, microRNA, physiology, regenerative medicine, stem cells 1 | BACKG ROU N D Ischemic disorders are the result of insufficiency in blood supply, leading to limited oxygen and nutrient transfer. Ischemia could involve most of the organs/tissues including the heart, brain, peripheral vessels, limbs, skin, retina, intestine and kidney. 1,2 Ischemic diseases are the leading cause of disability and mortality which impose an enormous burden on human healthcare systems worldwide. 3 Although current therapies for reperfusion including thrombolytic drugs, using vasodilator, 4,5 surgical bypass, and endovascular intervention, 6 have shown significant benefits in the treatment of the ischemic damage, however, these therapies often are not optimal for remodelling vascular beds, thereby ischemic diseases remain the leading cause of long-term disability. In addition, reperfusion has been found to be able to induce subsequent injury in ischemic tissue, a phenomenon termed ischemia-reperfusion (I/R) injury, which is a critical therapeutic challenge. 7 Besides, ischemia-induced and I/R-mediated injuries could lead to fibrosis and dysfunction of the damaged tissues in a long-time period. 8,9 Altogether, researchers have leaned towards finding the therapeutic strategies which could stimulate and enhance the regeneration of the ischemic tissues.
Ischemic disorders are contributed by vascular dysfunction, endothelial cell function impairment, vascular integrity deterioration and enhanced expression of adhesion molecules and inflammation mediators. 10 It is estimated that more than 500 million people worldwide will benefit from the treatment of the ischemic disorders. 11 Cell-based therapies are a revolutionary approach that have raised hopes for the treatment of ischemic disorders through various mechanisms such as angiogenesis induction, apoptosis inhibition and blocking inflammatory process. Stem cells are the most frequently used cells in cell-based therapies, with properties including differentiation capacity, self-renewal ability and secretion of beneficial paracrine factors. Stem cells have been shown to induce angiogenesis and provide blood supply in the ischemiadamaged organs. 12,13 Although cell-based therapies have shown promising results for treating various ischemic diseases, they are associated with multiple hindrances including low cell survival in the host's tissue and high expenses which emphasize the need for improving cell-based therapy strategies. 14 It has been shown that most transplanted cells could not survive more than 4 days posttransplantation. 15 It has been reported that <1% of systemic administered mesenchymal stem cells (MSCs) differentiate into the functional cells in the target tissue and a vast majority of them are trapped in the lung and liver. It is believed that transplanted cells exert their therapeutic effects and participate in angiogenesis via their paracrine activity. 16 Extracellular vesicles (EVs), main agents in cellular paracrine activity, are micro-and nano-sized vesicles which contain bioactive agents and are released by roughly all cells through fusion of multivesicular bodies with the plasma membrane and subsequent release to the intracellular space. Exosomes are a subgroup of EVs with an average size of 30-150 nm. [17][18][19] Exosomes are considered longrange intercellular communication tools that transfer various molecules including proteins, DNA, long non-coding RNAs (lncRNAs), message RNAs (mRNAs) and microRNAs (miRs) to the recipient cells.
Exosomal content represents the conditional and functional situation of the parent cell. 20,21 They easily pass vascular barriers (such as blood brain barrier) due to the nanoscale size and do not have any risk of tumorigenicity formation. Exosomes possess low immunogenicity as they lack the expression of major histocompatibility complex (MHC). Exosomes have a long-term storage capacity and could be stored at −20°C for months while their biological activity is preserved. 22,23 Exosomes are used as 'cell-free therapy' agents as they are responsible for the vast majority of cell-therapy-induced beneficent outcomes. Exosomes exert anti-apoptotic, anti-fibrotic, cell differentiation, immunomodulatory and pro-angiogenic effects. 24,25 It has been reported that the destruction of exosomes by ultrasonication abolishes cardiac progenitor cells (CPCs) angiogenic capacity, demonstrating the importance of exosomes in angiogenic induction. 26 It has been reported that the beneficial effects of endothelial progenitor cells (EPCs) in endothelial repair may greatly depend on their paracrine impacts in which exosomes play a central role in. 27 Better performance of CPCs transplantation compared with cardiospherederived cells (CDCs) transplantation in the treatment of myocardial infarction (MI) is mostly due to the greater angiogenesis induction of the CPC-derived exosomes (two-fold higher) and higher angiogenic capacity of miRNA cargo of CPC-derived exosomes. 20 In this review article, we discussed exosomes and their role in angiogenesis and highlighted recent application of them in ischemic disorders in preclinical models and clinical studies.  28,29 Besides the angiogenesis-associated cells, several pro-angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins, fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF) and hypoxia inaudible factor-1α (HIF-1α) play crucial roles in neovascularization. [30][31][32][33][34] Many prominent angiogenesis-associated factors such as proteins and nucleic acids are incorporated in exosomes, released by special cells and delivered to the recipient cells. Therefore, exosomal content and original cell types impressively affect the angiogenic potential of exosomes.

| E XOSOME: THE CELL OR , CONTENT AND MECHANIS M OF ANG IOG ENE S IS INDUC TION
Exosomes promote angiogenesis by transferring their content into recipient cells. The transferred molecules exert biochemical alterations in the recipient cell, leading to enhanced angiogenic activity. 35,36 Exosomes are considered 'mini-cells' because they contain multiple bioactive molecules according to their parent cell.
Considering the formation process of exosomes, their content is categorized into surface molecules and inner content. Same as a typical cell, the exosomal membrane consists of lipids, carbohydrates and proteins. however, cell-type-specific proteins are a wide range of exclusive proteins mediating various therapeutic and pathologic effects of exosomes. 37 Exosomes' pro-angiogenic content includes a variety of surface and internal molecules. More prominently, the internal proteins such as VEGF, angiopoietin-1 (Ang-1) and heat shock proteins (HSPs), as well as nucleic acids including miRNAs, lncRNAs and circular RNAs (circRNAs) participate in angiogenesis. 38,39 It is noteworthy that the internalization of exosomes by recipient cells is dependent on multiple factors, including exosome type and recipient cell type. For instance, it has been demonstrated that ECs and cardiac fibroblasts ingest MSC-derived exosomes with higher amounts compared with cardiomyocytes. Cardiomyocytes uptake EC-derived exosomes to a greater extent compared with MSCextracted exosomes, demonstrating the importance of exosome type. This phenomenon may be partially due to connexins and integrins inserted in the exosomal membrane from different cell types. 40 After administration of exosomes and subsequent entrance to systemic circulation, exosomes are distributed into tissues. 41 Following cellular uptake, the endocytic pathway results in breaking down of the exosomal cargo into their metabolites. 42 Kidneys, liver, spleen and lungs which possess a mononuclear phagocyte system, closely contribute to clearance of exosomes from circulation. 43 In vivo tracking of exosomes after administration by using sensitive, efficient and biocompatible methods and imaging techniques are highly desired to evaluate the pharmacokinetics of exosomes. 44 In this regard, pharmacokinetic analysis of gLuc-lactadherin labelled exosomes by bioluminescent imaging after intravenous injection demonstrated rapid clearance of exosomes with a half-life about 2 min. Also exosomes were mainly distributed to the liver followed by the lungs. 45 Consistently after 4 h of IV injection of I 125 labelled exosomes approximately 1.6%, 7% and 28% of the radioactivity was detected in the spleen, lungs and liver, respectively. 46 Exosomal content and composition depend on the original cell and the environmental condition. Various cells, including stem cells, mature cells, immune cells and tumour-associated cells, have been used to isolate exosomes for therapeutic angiogenesis. Stem cell-derived exosomes could significantly boost angiogenesis and re-establish blood supply when administered to ischemic areas. 47 Several stem cell sources have been administered regarding proangiogenic properties, such as MSCs, induced pluripotent stem cells (iPSCs), and adult progenitor cells. It is of crucial importance to note that some factors such as miR-20 or VEGF receptor-1 (VEGFR-1) demonstrate both pro-angiogenic and anti-angiogenic properties depending on the type of the recipient cell, dosage and microenvironment. [48][49][50][51] The pro-angiogenic content of exsosomes in addition to common cell sources is shown in Table 1.
Mechanism of exosome-induced angiogenesis could be categorized into three major activities: inducing pro-angiogenic factors and pathways, preserving the vascular network and regulating the inflammatory response.    Tube formation capacity of EPCs is impaired during MI due to C-X-C chemokine receptor type 7 (CXCR7) suppression. CXCR7 is a receptor of C-X-C motif chemokine 12 (CXCL12); CXCL12, also known as SDF-1, is a downstream target of Nrf2 and regulates EPCs migration to the ischemic region. Silent mating type information regulation 2 homologue 1 (SIRT1) activates Nrf2; It has been shown that exosomes derived from SIRT1-overexpressing adipose-derived MSCs (AD-MSCs) notably enhance EPCs' migration and tube formation through Nrf2 upregulation and subsequent CXCL12/CXCR7 signalling activation in EPCs. 63,64 Nuclear factor-κB improves angiogenesis through induction of VEGF expression. It has been reported that myocyte-derived exosomes stimulate the NF-κB pathway by inducing superoxide dismutase 2 (Sod2), probably via miR-130a transfer. Sod2 is a mitochondrial enzyme that protects the cell from oxidative stress via converting  Serpin E1 and homeobox proteins growth arrest A5 (HoxA5).

| Angiogenesis and related molecular pathways
Hampering these mediators and pathways finally results in the enhanced expression of angiogenic factors such as VEGF, Ang-1 and HIF-1α, as well as the upregulation of cell cycle proteins.
Considering VEGF as a crucial pro-angiogenic factor, preventing the inhibitors would increase the angiogenesis rate. PTEN is a potent angiostatic gene that suppresses the angiogenesis process It has been shown that miR-126 boosts angiogenesis through silencing sprouty-related EVH1 domain containing 1 (SPRED1) and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2), which results in RAF1/ERK1/2 upregulation and subsequent VEGF enhanced expression. 74,75 As with other proangiogenic factors, enhancing angiopoietin and HIF-1α has a desirable effect on angiogenesis. It has been revealed that miR-21-5p, which is abundant in endometrium-derived Taken together, exosomes could be an ideal tool for angiogenesis induction in ischemia-damaged tissues as they transfer proangiogenic factors into ECs and boost angiogenesis pathways and inhibit angiostatic signalling. In Figure 1, molecular mechanism underlying exosomess' pro-angiogenic activities are summarized. In brief, exosomes are able to diminish ischemic injury and preserve vascular network at the damaged site via inhibiting apoptosis, senescence and oxidative stress in recipient cells. Figure 2 demonstrates exosome's anti-apoptotic, anti-senescence and anti-oxidative mechanism of action.

| Exosomes and modulating inflammatory response
Following an ischemic insult, an inflammatory response is occurred in the damaged area due to the apoptosis, oxidative stress and the release of inflammatory cytokines. 102 The inflammatory response hampers the PI3K/Akt signalling, miR-126 silences ERRFI1, resulting in increased PI3K/Akt activity. p53 and Cdip1 facilitate apoptosis via triggering caspase-9; miR-125b and miR-21 enhance EC's survival via abolishing p53 and Cdip1 effects, respectively. ROS production which is enhanced by Nox2 and ERRFI1, causes oxidative stress, leading to EC senescence and blockage of EC's angiogenic capacity. Exosomal content can modify the senescence process of the EC and hence, improve angiogenesis. MiR-200a inhibits keap1, resulting in augmented Nrf2 anti-oxidative activity. ACE-2 blocks Nox2-mediated ROS production and miR-126 hampers ERRFI1 activity, both lead to decreased oxidative stress and cellular senescence In summary, exosome therapy could boost tissue regeneration and regulate inflammatory responses in ischemia-damaged tissues.
In Figure 3, immunomodulatory mechanisms of exosomes are shown. Local administration is described to be useful in several studies, but cannot be used for every organ. Also, the direct injection method seems more efficient in providing a sufficient amount of drug in the target tissue, however, it is more invasive and expensive than a systematic injection of exosomes, as it mostly requires special techniques. 115 The intramyocardial administration of the exosomes for treatment of cardiac disorders was used in in-vivo studies for the treatment of cardiologic disorders such as MI. 116

| MOD IFI C ATI ON S TR ATEG IE S TO IMPROVE E XOSOME FUN C TI ON
While exosome therapy has shown promising results as a contributory therapeutic tool for the treatment of ischemic disorders, the efficacy needs to be improved to make them an acceptable part of the therapeutic guidelines. Three main strategies that could promote exosome therapeutic potency are preconditioning, gene modification and bioconjugation are highlighted in this section.

| Preconditioning
Exosomes' content is extremely affected by the microenvironment in which their origin cells reside. 118 It has been demonstrated that through myocardial ischemia, cardiomyocytes' exosome production rate, as well as their exosomal pro-angiogenic content significantly increase. 119 Preconditioning is a method that simulates various microenvironments such as hypoxic microenvironment, acidic microenvironment, various physical stimuli and presence of diverse biologic and growth stimulations for the cells in order to promote their therapeutic and biogeneration aspects. 120 Different preconditioning strategies are available such as hypoxic preconditioning, physical preconditioning and preconditioning with drugs and chemicals. 121 In the setting of exosome generation, it has been shown that various preconditioning strategies could augment a cell's exosome biogenesis as well as enhancing the secreted exosomes' pro-angiogenic content. 122 Hypoxic preconditioning is the most utilized strategy in which cells undergo low oxygen tension in their culture milieu or cultured with hypoxia mimetic agents. 123 It has been demonstrated that mechanical stress with 15% static stretching could promote the production of exosomes enriched with miR-1246 pro-angiogenic factor from fibroblasts. 129 Taken together, preconditioning of parent cell is a cost-effective and efficient strategy to improve the quantity and biologic functions of the secreted exosomes. It is crucial to determine the most efficient preconditioning strategy for each cell type and biologic aspect which we intend to promote.

| Gene modification, protein and RNA transfection
Genetic modification is a cell manipulation strategy in which the target cell's genome is altered via using various techniques such as viral vectors resulting in DNA sequence alteration and subsequent upregulation or downregulation of specific genes. 130 As exosome biogenesis and content is proportionate to the parent cell, genetic modification of the parent cell alters its exosome biogenesis and content.
Viruses are appropriate tools for gene modification as they possess a natural instinct to infect the target cell's genome. 131

| Bioconjugation
In bioconjugation, specific biomolecules are included in exosomes. 64 A strategy to promote exosome targeting ability is conjugating target organ-specific ligands into the exosome's membrane. It has been shown that there are specific signal molecules on the surface of the exosomes that facilitate exosome uptake by specific tissues. 138

Conjugation of cyclo (Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)]
onto the exosome surface using bio-orthogonal copper-free click chemistry, improves its targeting capability since c(RGDyK) binds to integrin αvβ3 existing in reactive ECs in cerebral vascular network. It has been shown that c(RGDyK)-conjugated exosomes have a higher migration rate after IV administration to the ischemic brain. 139 Bioorthogonal chemistry method has also been used to conjugate IMTP with hypoxic preconditioned BM-MSC-derived exosomes and resulted in profoundly enhanced exosomal migration and retention into infarcted myocardium. 92 Ischemic regions have a low pH due to high glycolysis rate and low oxygen supply. 140 It has been demonstrated that conjugation of the intercalated motif (i-motif), a pHsensitive DNA strand enriched with cytosine, significantly promotes exosomes delivery to acidic areas, which could promote exosome targeted-delivery to ischemia-bearing sites. 141 In another study, hyaluronic acid grafted with 3-diethylamino propylamine (HDEA) was loaded into exosomes via sonication, a physical method to load cargos into exosomes via creating pores in the exosomal membrane by ultrasonic waves; it has been shown that membrane of HDEAloaded exosomes significantly desbalized in pH = 6.5, which resulted in releasing their content in an acidic environment. 142 Nanoscale cargos such as miRNAs could be loaded into the exosomes via incubation. It is possible to load miR-210 into the MSC-derived exosomes via cholesterol modification which creates lipophilic miRNAs that could efficiently emerge with exosome membrane; incubation of exosomes with lipophilic miR-210 enhances exosomal miR-210 level, which results in improved pro-angiogenic capacity. 143 Electroporation is another strategy to load biologic substances into the exosomes. Electroporation utilizes electrical flow to notch exosomal membrane in order to create micro-pores and enhance exosomes' permeability, thus facilitating molecular penetration into exosomes. It has been reported that electroporation of miR-132 into the MSCs-derived exosomes significantly enhances their angiogenesis induction in HUVECs. 23 Electroporation possesses a higher efficacy in loading cargos into the exosomes compared with incubation, but it is also associated with a higher risk for manipulating and disrupting the exosome' structure as well as a more complex procedure. 144 In Figure 4 different modification strategies to improve exosomes therapeutic capacity and their effects are shown.

| CLINI C AL APPLI C ATI ON OF E XOSOME S , A PER S PEC TIVE OVERVIE W
Although a huge amount of evidence has elucidated various therapeutic potentials of exosomes in ischemic disorders, there are several challenges in the safety and efficacy of exosome therapy which need to be overcome. 145  acute kidney injury (AKI). 158 In vivo studies regarding ischemic diseases were described in detail in Table 2.

TA B L E 2
In vivo studies of exosome applications for the treatment of ischemic disorders classified by disorders type, cell origin, mechanism of therapeutic action and route of delivery.  163 and colon cancer. 164 Table 3 summarized the published and ongoing studies regarding ischemic diseases.

| CON CLUS I ON AND FUTURE PER S PEC TIVE
Although exosomes have been extensively investigated as therapeutic modalities for ischemic diseases, there are still many challenges which need to be addressed, mainly in terms of optimization and improvement of isolation protocols and effective dose escalation.
One of the most important challenges that exosome-based therapies have faced is the rapid clearance and short half-life of exosomes in vivo. To produce practical scale of exosomes, scale-up in vitro cell culture systems should be established. This is a considerable challenge for health experts. A technique to enhance exosome production is application of three-dimensional (3D) culture system which supports better cell-to-cell communication and promotes exosome biogenesis. 165 It has been demonstrated that hUC-MSCs cultured in a 3D condition possess a 19.4 folds higher exosome production compared to the hUC-MSC cultured in 2D condition. 166 Moreover, exosomes could be used in combination with conventional therapies. For instance, integration of MSC-derived exosomes into scaffolds and hydrogels could significantly improve the wound healing process via promoting angiogenesis and inflammation regulation. 167 Exosome can also serve as an ideal vehicle for drug delivery. Various drugs as chemotherapeutics and angiogenesis-stimulators could be loaded on exosomes and delivered different biomedical components to target tissues with great efficacy. 168 As naked exosomes undergo extensive phagocytosis and clearance shortly after transplantation, it has been shown that embedding exosomes on biomaterials such as stents, cardiac patches and cell sheets could profoundly enhance their sustainability and therapeutic efficacy. 169 Tumour-associated exosomes contain considerable angiogenic molecules since angiogenesis is a crucial necessity for tumour development, expansion and far metastasis. It is known that cancerous cell-derived exosomes participate in tumour angiogenesis; thus, neoplastic cells could be suitable sources for angiogenic-exosome isolation. 68,170 Tumour cell characteristics exert an essential impact on the properties of secreted exosomes. For instance, chemoresistant ovarian cancer cell-derived exosomes possess more powerful angiogenic impacts than those derived from normal ovarian cancer cells. 171 Nevertheless, utilization of cancerous cells for exosome extraction may increase the risk of carcinogenesis in the target site. 172 In conclusion, exosomes could be an ideal therapeutic tools for the treatment of ischemic disorders due to their significant proangiogenic capacity and unique biological properties. However, for a prosperous clinical translation, it is crucial to optimize their therapeutic activity, define certain protocols for extraction, modification and administration, as well as conducting more investigations on their molecular mechanism of action.

ACK N O WLE D G E M ENTS
None.

FU N D I N G I N FO R M ATI O N
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.