Exosome: A Novel Approach to Stimulate Bone Regeneration through Regulation of Osteogenesis and Angiogenesis

The clinical need for effective bone regeneration therapy remains in huge demands. However, the current “gold standard” treatments of autologous and allogeneic bone grafts may result in various complications. Furthermore, safety considerations of biomaterials and cell-based treatment require further clarification. Therefore, developing new therapies with stronger osteogenic potential and a lower incidence of complications is worthwhile. Recently, exosomes, small vesicles of endocytic origin, have attracted attention in bone regeneration field. The vesicles travel between cells and deliver functional cargoes, such as proteins and RNAs, thereby regulating targeted cells differentiation, commitment, function, and proliferation. Much evidence has demonstrated the important roles of exosomes in osteogenesis both in vitro and in vivo. In this review, we summarize the properties, origins and biogenesis of exosomes, and the recent reports using exosomes to regulate osteogenesis and promote bone regeneration.     

Topical Application of Mesenchymal Stem Cell Exosomes Alleviates the Imiquimod Induced Psoriasis-Like Inflammation

Severe psoriasis, a chronic inflammatory skin disease is increasingly being effectively managed by targeted immunotherapy but long-term immunotherapy poses health risk and loss of response. Therefore, there is a need for alternative therapy strategies. Mesenchymal stem/stromal cell (MSC) exosomes are widely known for their potent immunomodulatory properties. Here we investigated if topically applied MSC exosomes could alleviate psoriasis-associated inflammation. Topically applied fluorescent exosomes on human skin explants were confined primarily to the stratum corneum with <1% input fluorescence exiting the explant over a 24-h period. Nevertheless, topically applied MSC exosomes in a mouse model of imiquimod (IMQ) psoriasis significantly reduced IL-17 and terminal complement activation complex C5b-9 in the mouse skin. MSC exosomes were previously shown to inhibit complement activation, specifically C5b-9 complex formation through CD59. Infiltration of neutrophils into the stratum corneum is characteristic of psoriasis and neutrophils are a major cellular source of IL-17 in psoriasis through the release of neutrophil extracellular traps (NETs). We propose that topically applied MSC exosomes inhibit complement activation in the stratum corneum and this alleviates IL-17 release by NETS from neutrophils that accumulate in and beneath the stratum corneum.     

Potential of Exosomes for Diagnosis and Treatment of Joint Disease: Towards a Point-of-Care Therapy for Osteoarthritis of the Knee

In the knee joint, articular cartilage injury can often lead to osteoarthritis of the knee (OAK). Currently, no point-of-care treatment can completely address OAK symptoms and regenerate articular cartilage to restore original functions. While various cell-based therapies are being developed to address OAK, exosomes containing various components derived from their cells of origin have attracted attention as a cell-free alternative. The potential for exosomes as a novel point-of-care treatment for OAK has been studied extensively, especially in the context of intra-articular treatments. Specific exosomal microRNAs have been identified as possibly effective in treating cartilage defects. Additionally, exosomes have been studied as biomarkers through their differences in body fluid composition between joint disease patients and healthy subjects. Exosomes themselves can be utilized as a drug delivery system through their manipulation and encapsulation of specific contents to be delivered to specific cells. Through the combination of exosomes with tissue engineering, novel sustained release drug delivery systems are being developed. On the other hand, many of the functions and activities of exosomes are unknown and challenges remain for clinical applications. In this review, the possibilities of intra-articular treatments utilizing exosomes and the challenges in using exosomes in therapy are discussed.     

Exosomal Vimentin from Adipocyte Progenitors Protects Fibroblasts against Osmotic Stress and Inhibits Apoptosis to Enhance Wound Healing

Mechanical stress following injury regulates the quality and speed of wound healing. Improper mechanotransduction can lead to impaired wound healing and scar formation. Vimentin intermediate filaments control fibroblasts’ response to mechanical stress and lack of vimentin makes cells significantly vulnerable to environmental stress. We previously reported the involvement of exosomal vimentin in mediating wound healing. Here we performed in vitro and in vivo experiments to explore the effect of wide-type and vimentin knockout exosomes in accelerating wound healing under osmotic stress condition. Our results showed that osmotic stress increases the size and enhances the release of exosomes. Furthermore, our findings revealed that exosomal vimentin enhances wound healing by protecting fibroblasts against osmotic stress and inhibiting stress-induced apoptosis. These data suggest that exosomes could be considered either as a stress modifier to restore the osmotic balance or as a conveyer of stress to induce osmotic stress-driven conditions.     

Emerging Roles of Exosomes in Huntington’s Disease

Huntington’s disease (HD) is a rare hereditary autosomal dominant neurodegenerative disorder, which is caused by expression of mutant huntingtin protein (mHTT) with an abnormal number of glutamine repeats in its N terminus, and characterized by intracellular mHTT aggregates (inclusions) in the brain. Exosomes are small extracellular vesicles that are secreted generally by all cell types and can be isolated from almost all body fluids such as blood, urine, saliva, and cerebrospinal fluid. Exosomes may participate in the spreading of toxic misfolded proteins across the central nervous system in neurodegenerative diseases. In HD, such propagation of mHTT was observed both in vitro and in vivo. On the other hand, exosomes might carry molecules with neuroprotective effects. In addition, due to their capability to cross blood-brain barrier, exosomes hold great potential as sources of biomarkers available from periphery or carriers of therapeutics into the central nervous system. In this review, we discuss the emerging roles of exosomes in HD pathogenesis, diagnosis, and therapy.     

Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats

Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91^phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.     

Exosomes Derived from Mesenchymal Stem Cells

The functional mechanisms of mesenchymal stem cells (MSCs) have become a research focus in recent years. Accumulating evidence supports the notion that MSCs act in a paracrine manner. Therefore, the biological factors in conditioned medium, including exosomes and soluble factors, derived from MSC cultures are being explored extensively. The results from most investigations show that MSC-conditioned medium or its components mediate some biological functions of MSCs. Several studies have reported that MSC-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage, suppressing inflammatory responses, and modulating the immune system. However, the mechanisms are still not fully understood and the results remain controversial. Compared with cells, exosomes are more stable and reservable, have no risk of aneuploidy, a lower possibility of immune rejection following in vivo allogeneic administration, and may provide an alternative therapy for various diseases. In this review, we summarize the properties and biological functions of MSC-derived exosomes and discuss the related mechanisms.     

Circulating Exosomal miR-221 from Maternal Obesity Inhibits Angiogenesis via Targeting Angptl2

Maternal obesity disrupts both placental angiogenesis and fetus development. However, the links between adipocytes and endothelial cells in maternal obesity are not fully understood. The aim of this study was to characterize exosome-enriched miRNA from obese sow’s adipose tissue and evaluate the effect on angiogenesis of endothelial cells. Plasma exosomes were isolated and analyzed by nanoparticle tracking analysis (NTA), electron morphological analysis, and protein marker expression. The number of exosomes was increased as the gestation of the sows progressed. In addition, we found that exosomes derived from obese sows inhibited endothelial cell migration and angiogenesis. miRNA detection showed that miR-221, one of the miRNAs, was significantly enriched in exosomes from obese sows. Further study demonstrated that exosomal miR-221 inhibited the proliferation and angiogenesis of endothelial cells through repressing the expression of Angptl2 by targeting its 3′ untranslated region. In summary, miR-221 was a key component of the adipocyte-secreted exosomal vesicles that mediate angiogenesis. Our study may be a novel mechanism showing the secretion of “harmful” exosomes from obesity adipose tissues causes placental dysplasia during gestation.     

Expression Profiling of Exosomal miRNAs Derived from Human Esophageal Cancer Cells by Solexa High-Throughput Sequencing

Cellular genetic materials, such as microRNAs (miRNAs), mRNAs and proteins, are packaged inside exosomes, small membrane vesicles of endocytic origin that are released into the extracellular environment. These cellular genetic materials can be delivered into recipient cells, where they exert their respective biological effects. However, the miRNA profiles and biological functions of exosomes secreted by cancer cells remain unknown. The present study explored the miRNA expression profile and distribution characteristics of exosomes derived from human esophageal cancer cells through Solexa high-throughput sequencing. Results showed that 56,421 (2.94%) unique sequences in cells and 7727 (0.63%) in exosomes matched known miRNAs. A total of 342 and 48 known miRNAs were identified in cells and exosomes, respectively. Moreover, 64 and 32 novel miRNAs were predicted in cells and exosomes, respectively. Significant differences in miRNA expression profiles were found between human esophageal cancer cells and exosomes. These findings provided new insights into the characteristics of miRNAs in exosomes derived from human esophageal cancer cells and the specific roles of miRNAs in intercellular communication mediated by exosomes in esophageal cancer.     

Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.     

The Role of Melanoma Cell-Derived Exosomes (MTEX) and Photodynamic Therapy (PDT) within a Tumor Microenvironment

Photodynamic Therapy (PDT), an unconventional cancer therapy with optimistic desirable effects, utilizes the delivery of a photosensitizer (PS) that is activated by light at a particular wavelength and inducing oxidative cytotoxic damage of a tumor and its surrounding vasculature. Deeper seated tumors such as internally metastasized melanomas are more difficult to treat with PDT as the penetration of laser light to those sites is less. Limitations in targeting melanomas can also be attributed to melanin pigments that hinder laser light from reaching targeted sites. Exosomes serve as naturally occurring nanoparticles that can be re-assembled with PSs, improving targeted cellular absorption of photosensitizing agents during PDT. Additionally, studies indicate that exosomes released from PDT-treated tumor cells play a critical role in mediating anti-tumor immune responses. This review collates the role of Melanoma Cell-Derived Exosomes (MTEX) in immune response mediation and metastasis. Tumor Cell-Derived Exosomes (TEX) post PDT treatment are also reviewed, as well as the effects of exosomes as carriers of photosensitizers and delivery systems for PDT. The understanding and research on the role of melanoma exosomes induced by Photodynamic Therapy and their tumor microenvironment will assist in future research in treatment prospects and implications.     

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.     

Targeting the Redox Balance in Inflammatory Skin Conditions

Reactive oxygen species (ROS) can be both beneficial and deleterious. Under normal physiological conditions, ROS production is tightly regulated, and ROS participate in both pathogen defense and cellular signaling. However, insufficient ROS detoxification or ROS overproduction generates oxidative stress, resulting in cellular damage. Oxidative stress has been linked to various inflammatory diseases. Inflammation is an essential response in the protection against injurious insults and thus important at the onset of wound healing. However, hampered resolution of inflammation can result in a chronic, exaggerated response with additional tissue damage. In the pathogenesis of several inflammatory skin conditions, e.g., sunburn and psoriasis, inflammatory-mediated tissue damage is central. The prolonged release of excess ROS in the skin can aggravate inflammatory injury and promote chronic inflammation. The cellular redox balance is therefore tightly regulated by several (enzymatic) antioxidants and pro-oxidants; however, in case of chronic inflammation, the antioxidant system may be depleted, and prolonged oxidative stress occurs. Due to the central role of ROS in inflammatory pathologies, restoring the redox balance forms an innovative therapeutic target in the development of new strategies for treating inflammatory skin conditions. Nevertheless, the clinical use of antioxidant-related therapies is still in its infancy.     

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.     

Nitric Oxide and Reactive Oxygen Species in the Pathogenesis of Preeclampsia

Preeclampsia (PE) is characterized by disturbed extravillous trophoblast migration toward uterine spiral arteries leading to increased uteroplacental vascular resistance and by vascular dysfunction resulting in reduced systemic vasodilatory properties. Its pathogenesis is mediated by an altered bioavailability of nitric oxide (NO) and tissue damage caused by increased levels of reactive oxygen species (ROS). Furthermore, superoxide (O₂⁻) rapidly inactivates NO and forms peroxynitrite (ONOO⁻). It is known that ONOO⁻ accumulates in the placental tissues and injures the placental function in PE. In addition, ROS could stimulate platelet adhesion and aggregation leading to intravascular coagulopathy. ROS-induced coagulopathy causes placental infarction and impairs the uteroplacental blood flow in PE. The disorders could lead to the reduction of oxygen and nutrients required for normal fetal development resulting in fetal growth restriction. On the other hand, several antioxidants scavenge ROS and protect tissues against oxidative damage. Placental antioxidants including catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protect the vasculature from ROS and maintain the vascular function. However, placental ischemia in PE decreases the antioxidant activity resulting in further elevated oxidative stress, which leads to the appearance of the pathological conditions of PE including hypertension and proteinuria. Oxidative stress is defined as an imbalance between ROS and antioxidant activity. This review provides new insights about roles of oxidative stress in the pathophysiology of PE.     

Enhanced Nerve Regeneration by Exosomes Secreted by Adipose-Derived Stem Cells with or without FK506 Stimulation

Exosomes secreted by adipose-derived stem cells (ADSC-exo) reportedly improve nerve regeneration after peripheral nerve injury. Herein, we investigated whether pretreatment of ADSCs with FK506, an immunosuppressive drug that enhances nerve regeneration, could secret exosomes (ADSC-F-exo) that further augment nerve regeneration. Designed exosomes were topically applied to injured nerve in a mouse model of sciatic nerve crush injury to assess the nerve regeneration efficacy. Outcomes were determined by histomorphometric analysis of semi-thin nerve sections stained with toluidine blue, mouse neurogenesis PCR array, and neurotrophin expression in distal nerve segments. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile potential exosomal proteins facilitating nerve regeneration. We observed that locally applied ADSC-exo and ADSC-F-exo significantly enhanced nerve regeneration after nerve crush injury. Pretreatment of ADSCs with FK506 failed to produce exosomes possessing more potent molecules for enhanced nerve regeneration. Proteomic analysis revealed that of 192 exosomal proteins detected in both ADSC-exo and ADSC-F-exo, histone deacetylases (HDACs), amyloid-beta A4 protein (APP), and integrin beta-1 (ITGB1) might be involved in enhancing nerve regeneration.     

A Soluble Receptor for Advanced Glycation End-Products Inhibits Hypoxia/Reoxygenation-Induced Apoptosis in Rat Cardiomyocytes via the Mitochondrial Pathway

Severe myocardial dysfunction and tissue damage resulting from ischemia/reperfusion (I/R) is a common clinical scenario in patients with certain types of heart diseases and therapies such as thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting, and cardiac transplantation. The underlining mechanism of endogenous cardiac protection after I/R injury has been a focus of current research. Growing evidences suggests that soluble receptor for advanced glycation end-products (sRAGE) has a cardioprotective effect; however, its role in I/R injury remains unclear. We hypothesized that exogenous administration of sRAGE during hypoxia/reoxygenation (H/R) induces cardioprotection by inhibiting cardiomyocyte apoptosis via multiple signals, involving mitochondrial membrane potential (MMP), the mitochondrial permeability transition pore (mPTP), mitochondrial cytochrome c, caspase-3, Bcl-2 and Bax. Neonatal rat cardiomyocytes underwent hypoxia for 3-h followed by 2-h reoxygenation or were treated with sRAGE for 10 min before H/R. Compared with H/R alone, sRAGE pretreatment reduced H/R-induced cardiomyocyte apoptosis from 27.9% ± 5.9% to 9.4% ± 0.7% (p < 0.05). In addition, sRAGE treatment significantly inhibited H/R-induced mitochondrial depolarization and mPTP opening, reduced mitochondrial cytochrome c leakage, caspase-3 and caspase-9 activity, and decreased the ratio of Bax to Bcl-2. Therefore, we conclude that the exogenous administration of sRAGE during H/R is involved in cardioprotection by inhibiting apoptosis via the mitochondrial pathway, which, if further confirmed in vivo, may have important clinical implications during H/R.     

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.     

Combinational Therapy of Cardiac Atrial Appendage Stem Cells and Pyridoxamine: The Road to Cardiac Repair?

Myocardial infarction (MI) occurs when the coronary blood supply is interrupted. As a consequence, cardiomyocytes are irreversibly damaged and lost. Unfortunately, current therapies for MI are unable to prevent progression towards heart failure. As the renewal rate of cardiomyocytes is minimal, the optimal treatment should achieve effective cardiac regeneration, possibly with stem cells transplantation. In that context, our research group identified the cardiac atrial appendage stem cells (CASCs) as a new cellular therapy. However, CASCs are transplanted into a hostile environment, with elevated levels of advanced glycation end products (AGEs), which may affect their regenerative potential. In this study, we hypothesize that pyridoxamine (PM), a vitamin B6 derivative, could further enhance the regenerative capacities of CASCs transplanted after MI by reducing AGEs’ formation. Methods and Results: MI was induced in rats by ligation of the left anterior descending artery. Animals were assigned to either no therapy (MI), CASCs transplantation (MI + CASCs), or CASCs transplantation supplemented with PM treatment (MI + CASCs + PM). Four weeks post-surgery, global cardiac function and infarct size were improved upon CASCs transplantation. Interstitial collagen deposition, evaluated on cryosections, was decreased in the MI animals transplanted with CASCs. Contractile properties of resident left ventricular cardiomyocytes were assessed by unloaded cell shortening. CASCs transplantation prevented cardiomyocyte shortening deterioration. Even if PM significantly reduced cardiac levels of AGEs, cardiac outcome was not further improved. Conclusion: Limiting AGEs’ formation with PM during an ischemic injury in vivo did not further enhance the improved cardiac phenotype obtained with CASCs transplantation. Whether AGEs play an important deleterious role in the setting of stem cell therapy after MI warrants further examination.