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Review
. 2021 Jun 25;10(7):1596.
doi: 10.3390/cells10071596.

Extracellular Vesicles in Organ Fibrosis: Mechanisms, Therapies, and Diagnostics

David R Brigstock  1   2
Affiliations
Review

Extracellular Vesicles in Organ Fibrosis: Mechanisms, Therapies, and Diagnostics

David R Brigstock. Cells. .

Abstract

Fibrosis is the unrelenting deposition of excessively large amounts of insoluble interstitial collagen due to profound matrigenic activities of wound-associated myofibroblasts during chronic injury in diverse tissues and organs. It is a highly debilitating pathology that affects millions of people globally and leads to decreased function of vital organs and increased risk of cancer and end-stage organ disease. Extracellular vesicles (EVs) produced within the chronic wound environment have emerged as important vehicles for conveying pro-fibrotic signals between many of the cell types involved in driving the fibrotic response. On the other hand, EVs from sources such as stem cells, uninjured parenchymal cells, and circulation have in vitro and in vivo anti-fibrotic activities that have provided novel and much-needed therapeutic options. Finally, EVs in body fluids of fibrotic individuals contain cargo components that may have utility as fibrosis biomarkers, which could circumvent current obstacles to fibrosis measurement in the clinic, allowing fibrosis stage, progression, or regression to be determined in a manner that is accurate, safe, minimally-invasive, and conducive to repetitive testing. This review highlights the rapid and recent progress in our understanding of EV-mediated fibrotic pathogenesis, anti-fibrotic therapy, and fibrosis staging in the lung, kidney, heart, liver, pancreas, and skin.

Keywords: collagen; exosome; extracellular matrix; extracellular vesicle; fibrogenic; fibrosis; myofibroblast.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Principal cellular events leading to fibrosis. Injury to epithelial cells results in their release of pro-inflammatory cytokines, which is a stimulus for infiltration of macrophages and immune cells to the injury site. This results in an inflammatory environment in which cytokines, chemokines, and growth factors are produced that drive the production and action of contractile αSMA-producing myofibroblasts. Myofibroblasts arise by activation of resident fibroblasts or circulating fibrocytes or are the result of transdifferentiation from other cell types such as epithelial cells, endothelial cells, or pericytes. In acute injury, myofibroblasts transiently produce ECM components such as collagen, laminin, and FN which are necessary for normal wound healing, and parenchymal repopulation. During persistent or recurrent episodic injury, the inflammatory phase is protracted leading to unrelenting myofibroblastic activity, which is manifested as excessive production of ECM components that are deposited in the interstitial space as scar material, or fibrosis.
Figure 2
Figure 2
Pathways of EV biogenesis and action. Three type of EVs are produced by most cells. Exosome biogenesis is initiated by the involution and pinching off of the endosomal membrane resulting in the production of an ILV that contains cytoplasmic constituents. As ILVs accumulate, MVBs are generated, which are then either degraded via the lysosomal pathway or trafficked to the cell surface whereupon they fuse with plasma membrane and liberate their contents, now becoming exosomes, into the extracellular space. Mircovesicles also contain similar cytoplasmic constituents but are generated by fission of the plasma membrane. Apoptotic bodies contain components of cell degradation and form by cytoplasmic bulging and separation from the cell as a result of cytosketetal breakdown during cell disassembly. Microvesicles and exosomes may bind to ECM components in the interstitial space or may be internalized by target cells, either by fusion with the plasma membrane or by endocytosis, both of which result in delivery of their respective molecular payloads into the recipient cell. Once released into the extracellular space, EVs may alternatively be carried in interstitial fluids into the main body fluids allowing them to target cells at distant sites or to be cleared.
Figure 3
Figure 3
Proposed EV pathways in the pathogenesis of pulmonary fibrosis. Prolonged alveolar injury results in the production and interstitial deposition of type I collagen by αSMA-positive myofibroblasts and the resulting increased edema and expansion of fibrotic ECM impinges on alveolar air space, structure and function, severely limiting gaseous exchange and decreasing lung performance. Myofibroblast expansion is the result of transdifferentiation/activation of resident interstitial fibroblasts, bone marrow-derived fibrocytes, endothelial cells, epithelial cells, or pericytes. Altered EV components such as Wnt5a and let-7d from these cell types are proposed to promote the transition into myofibroblasts and/or to enhance activation and fibrogenesis in the accumulating myofibroblast population through the regulation of TGF-β, Smad and β-catenin pathways, while increased EV PD-L1 dampens T cell responses and promotes fibroblast migration. Further, miR-23b-3p and miR-494-3p in EVs from activated fibroblasts suppress notch signaling and drives cell senescence in epithelial cells. EVs from infiltrating or activated M2 macrophages contain altered levels of miRs-328 and -125a-5p which drive fibroblast transdifferentiation and collagen production in fibroblasts, and this process is exacerbated by the effect of 1L-10-enriched EVs from TNF-α-primed neutrophils. Pro-fibrogenic EVs in BALF originate from damaged, infiltrating, and activated cells in lung tissue and edema fluid but in most studies the precise cellular sources of BALF EVs have not been definitively determined. Transdifferentiation of pericytes is triggered as a response to EVs from BALF or capillary endothelial cells which contain, respectively, suppressed let-7d or miR-107. Only cells with a demonstrated role in EV production or response are shown; some of the depicted EV pathways are surmised from in vitro observations and have not been demonstrated in vivo. See text for details.
Figure 4
Figure 4
Proposed EV pathways in the pathogenesis of renal fibrosis. EVs are produced in enhanced numbers by stressed or injured TEC downstream of Shh and they cause proliferation and fibrogenesis in fibroblasts, in part by downregulation of SOCS2 by EV miR-196b-5p. TEC EVs also drive EMT and activation in TEC themselves via processes that are dependent on the action of EV TGF-β, transglutaminase 2, or miR-21, the latter of which is associated with regulation of PTEN/AKT or PPAR/HIF in recipient cells. Podocyte injury and drop-out is a feature of DN and EVs from these cells are present in urine and, when produced in the context of DN-like high glucose conditions, drive ECM production via a TGF-β/p38/Smad axis in TEC and mesangial cells. High glucose levels also result in the production of EVs by GEC that drive Wnt/β-catenin-mediated EMT in podocytes or circular RNA-mediated EMT in mesangial cells. Macrophages exposed to high glucose produce pro-inflammatory EVs that are themselves macrophage-activating and further stimulate TGF-β-dependent activation of mesangial cells. Production by mesangial cells of pro-fibrogenic and pro-pathogenic molecules such as FN and Ang II is stimulated by EVs that are produced by mesangial cells exposed to high glucose concentrations. Only cells with a demonstrated role in EV production or response are shown; some of the depicted EV pathways are deduced from in vitro observations and have not been demonstrated in vivo. See text for details.
Figure 5
Figure 5
Proposed EV pathways in the pathogenesis of cardiac fibrosis. Cardiomyocytes exposed to stress such as hypoxia produce EVs that promote mesenchymal transition, survival, and fibrogenesis in cardiac fibroblasts/myofibroblasts in part due to delivery of enriched EV cargo components that include Neat 1, miR-29b-3p, miR-30d-5p, miR-100-5p, miR181a, miR-21 and miR-208a, the latter two of which target, respectively, AKT-PTEN and Dyrk2. TNF-α-stimulated cardiomyocytes and cardiac fibroblasts produce EVs that are exchanged between them and which can promote increased oxidative stress by delivery of Nrf2-tartgeting miR-27a, -28-3p, or -34a, while profibrotic pathways in cardiac fibroblasts are promoted by delivery of fibroblast EVs enriched with Wnt3a or Wnt5a. High glucose-stimulated macrophages produce EVs that are enriched in HuR which is required for EV-stimulated expression of inflammatory or fibrogenic genes in cardiac fibroblasts. Macrophage EVs are also enriched in miR-155 which exerts, firstly, anti-proliferative and pro-inflammatory effects in cardiac fibroblasts by targeting Son of Sevenless 1/SOCS1 and, secondly, pyroptosis, hypertrophy and fibrosis in cardiomyocytes by its targeting of Foxo3a. Finally, age-related declines in HSP70 levels in serum EVs are associated with increased cardiac fibroblast proliferation. Only cells with a demonstrated role in EV production or response are shown; some of the depicted EV pathways are deduced from in vitro observations and have not been demonstrated in vivo. See text for details.
Figure 6
Figure 6
Proposed EV pathways in the pathogenesis of hepatic fibrosis. Hepatocytes that are injured by exposure to hepatitis viruses, alcohol, or free fatty acids produce increased numbers of EVs due to activation of EV biogenic components (e.g., ROCK-1) and suppression of autophagy-associated late endosomes. These EVs drive activation and function in macrophages or HSC, or alternatively are released into the circulation. Production by macrophages of pro-inflammatory cytokines (e.g., IL-1β, IL-6) occurs upon interactions of hepatocyte EVs with TLR and DR5 as well as stimulation of NF-κB-activated NLRP3 inflammasomes. Hepatocyte EVs also stimulate TLR3 expression in HSC which causes HSC activation and drives an IL-17A positive feedback loop between HSC and δ T cells that exacerbates inflammation and fibrosis. HSC activation and fibrogenesis is directly stimulated by hepatocyte EVs and involves various mechanisms such regulation of PPARγ by EV miR-128-3p, -27b, -130b, Smad 7 by EV miR-27a, Nrld2 by miR-181, SOCS3 by EV miR-19a, TGF-β by miR-192, and miR-26b and its collagen 1α2 target by EV MALAT. HSC themselves release EVs that are enriched in PDGFα, bind to HSC integrins and heparan sulfate proteoglycans, and stimulate HSC migration, activation, and fibrogenesis. EVs produced downstream of activation of apoptosis signaling regulating kinase 1 or HIF-1 during HSC activation deliver GLUT1 and PKM2 to quiescent HSC, KC or LSECs and are pro-fibrogenic in part due to suppressed EV miR-30a levels and a proteomic cargo that is enriched for ECM-, proteasome- and collagen-associated components. AKT activation and migration in HSC is stimulated by EVs from liver sinusoidal endothelial cells while vascular endothelial cells undergo tube formation in response to VEGF-enriched EVs from fibrotic myofibroblasts and demonstrate enhanced adhesion to monocytes in the presence of integrin- β1-enriched EVs from lipotoxic hepatocytes. Activated macrophages produce miR-103-3p-eriched EVs that stimulate HSC activation and fibrogenesis via suppression of KLF4. EVs in serum or plasma from fibrotic patients activate HSC and are likely derived from injured hepatocytes (e.g., NAFLD, alpha-1 antitrypsin deficiency) or mast cells (e.g., systemic mastocytosis). Only cells with a demonstrated role in EV production or response are shown; some of the depicted EV pathways are deduced from in vitro observations and have not been demonstrated in vivo. See text for details.
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