The uPA/uPAR System Orchestrates the Inflammatory Response, Vascular Homeostasis, and Immune System in Fibrosis Progression

Fibrotic diseases, such as systemic sclerosis (SSc), idiopathic pulmonary fibrosis, renal fibrosis and liver cirrhosis are characterized by tissue overgrowth due to excessive extracellular matrix (ECM) deposition. Fibrosis progression is caused by ECM overproduction and the inhibition of ECM degradation due to several events, including inflammation, vascular endothelial dysfunction, and immune abnormalities. Recently, it has been reported that urokinase plasminogen activator (uPA) and its receptor (uPAR), known to be fibrinolytic factors, orchestrate the inflammatory response, vascular homeostasis, and immune homeostasis system. The uPA/uPAR system may show promise as a potential therapeutic target for fibrotic diseases. This review considers the role of the uPA/uPAR system in the progression of fibrotic diseases.


The Role of the uPA/uPAR System in Fibrosis
Fibrosis is characterized by ECM deposition due to the overproduction of ECM and the inhibition of ECM degradation. Many fibrotic events, such as vascular endothelial dysfunction and immune abnormalities, are associated with the activation and differentiation of myofibroblasts and the inhibition of ECM-depredating proteases, including MMPs and plasmin, which cause ECM deposition [9,11,[64][65][66][67].
Plasmin not only degrades fibrin but also induces MMP and PAR activity. Fibrin and PAR activation promotes fibrosis, and MMPs play an important role in ECM degradation [76,77]. In addition, plasmin-induced hepatocyte growth factor (HGF) activation and vascular endothelial growth factor (VEGF) release may affect fibrosis progression [78,79]. Furthermore, α2AP and PAI-1 deficiency attenuated fibrosis progression in fibrosis model mice [21,78,[80][81][82]. These data suggest that the uPA/uPAR system plays a pivotal role in the progression of fibrosis through multiple plasmin-dependent and plasmin-independent mechanisms.
These uPAR-associated factors regulate the activation and differentiation of myofibroblasts, suggesting that the interaction with uPAR may play an important role in myofibroblast differentiation.

Other Fibrinolytic Factors and Myofibroblasts
The uPA/uPAR system, as well as other fibrinolytic factors, such as tPA, PAI-1 and α2AP are associated with myofibroblast activation and differentiation.
tPA is a Plg activator that also converts Plg into plasmin. tPA deficiency increases apoptosis of interstitial myofibroblasts in a mouse model of obstructive injury, and tPA is associated with myofibroblast apoptosis [127]. In addition, tPA induces myofibroblast activation through LRP-1 activation [128].
α2AP rapidly inactivates plasmin by inducing the formation of plasmin-α2AP complex. In contrast, α2AP deficiency attenuates dermal fibrosis in mice [80]. In addition, α2AP promotes myofibroblast differentiation and fibrosis through ATGL activation [136], and the blockade of α2AP by neutralizing antibodies or miRNA attenuates myofibroblast differentiation and fibrosis [22,137]. Furthermore, α2AP is associated with the induction of EMT and EndoMT [21,138]. α2AP may also regulate myofibroblast activation and differentiation through multiple functions.

Suppression of ECM Depredating Protease in Fibrosis
The suppression of ECM degradation, as well as the overproduction of ECM, causes the progression of fibrosis [10]. Proteases, including MMPs, plasmin, and uPA, regulate ECM degradation, and the activity of these proteases is inhibited by TIMPs, α2AP, and PAI-1, respectively. An imbalance between proteases and anti-proteases may promote fibrosis progression.

The Role of the uPA/uPAR System in EC Functions
Plasmin is known to play an important role on the maintenance of the vascular endothelial function through fibrinolysis, MMP and cytokine activation, and ECM degradation [11,158]. In addition, plasmin regulates fibrin-, MMP-, and cytokine-mediated EC proliferation, migration, and apoptosis [159][160][161]. In contrast, uPA protects ECs from apoptosis [162,163], and promotes EC proliferation [164]. The proteolytically inactive recombinant of uPA inhibits EC migration [165], and uPAR deficiency alters EC functions, including adhesion, migration, proliferation, and capillary tube formation, and decreases angiogenic functions [166]. uPAR antagonist inhibits the motility of ECs [167]. In addition, the uPA/uPAR system cross-talks with integrins and VEGFR2 and mediates EC tube formation [168]. Furthermore, uPAR-integrin interaction regulates EC migration [169], and the uPA/uPAR system is associated with growth factor-induced EC migration [170,171]. The uPA/uPAR system may regulate EC functions through plasmin-dependent or plasminindependent mechanisms.

The Role of the uPA/uPAR System in Angiogenesis
uPA regulates plasmin production and plays an important role in angiogenesis [27]. PA regulates the VEGFR1 and VEGFR2 expression by binding to haematopoietically expressed homeobox protein (HHEX) transcription factor and mediates angiogenesis [172]. uPA and uPAR shRNA enhance TIMP-1-mediated soluble VEGFR1 (sVEGFR1) secretion, and inhibit angiogenesis [173]. Plasmin regulates the vascular endothelial function through fibrinolysis, ECM degradation, and activation of growth factors [11]. In addition, plasmin can release pro-angiogenic factor VEGF from ECM and plays an important role in angiogenesis [174]. The deficiency of α2AP promotes angiogenesis through VEGF over-release in the woundhealing process [79], and α2AP causes the impairment of VEGF signaling [175].

The Role of the uPA/uPAR System in Coagulation
Hypercoagulation involving fibrin formation contributes to fibrosis progression [77]. The uPA/uPAR system plays a pivotal role in fibrin degradation through plasmin production. uPA and plasmin inhibitors, PAI-1 and α2AP, are elevated in several fibrotic tissue types [11], and the increase in the expression of these factors may cause the impairment of fibrinolysis through the direct inhibition of uPA and plasmin. The impairment of fibrinolysis causes fibrosis, and improvement in fibrinolysis restores fibrosis [77]. In addition, fibrin degradation product fragment can potentiate TGF-β-induced myofibroblast formation [181]. An imbalance in coagulation and fibrinolysis may thus play a critical role in fibrosis progression.

The Role of the uPA/uPAR System in Vascular Tone Alteration and Hypertension
Hypertension causes cardiac fibrosis [182], while fibrosis leads to pulmonary hypertension [183]. The balance between vasoconstrictor and vasodilator mediators, such as nitric oxide (NO), prostacyclin (PGI2), and endothelin-1 (ET-1), regulates vascular tone, and a shift toward vasoconstriction is associated with hypertension progression [184]. The ratio of uPA and PAI-1 is decreased in idiopathic pulmonary fibrosis patients with pulmonary hypertension [185]. uPA deficiency attenuates hypoxia-induced pulmonary arterial hypertension (PAH) progression [186]. uPAR is associated with SSc-associated PAH [187]. NO is a major vasodilation mediator, and is produced by NO synthase (NOS, including endothelial NOS [eNOS] and inducible NOS [iNOS]). uPA induces eNOS activation through LRP [188]. Treatment with UPARANT, which inhibits uPAR binding to the FPR, attenuates iNOS and NO production [189]. In contrast, iNOS and NO downregulate uPAR expression under hypoxic conditions [190]. The uPA/uPAR system may be associated with vasodilation and the onset of hypertension.

Immune Abnormalities and Inflammation in Fibrosis
Chronic inflammation leads to excessive tissue repair and triggers fibrosis progression [191]. Various stimuli, such as tissue injury, allergic response, autoimmune conditions, and infection, can cause inflammation and recruit and activate immune cells [192]. Immune cells, including T-cells, B-cells, macrophages, and dendritic cells (DCs), have been observed in fibrotic tissues [191,193]. T-cells (Th1 cells, Th2 cells, Th17 cells, and regulatory T-cells) secrete various cytokines, including IFN-γ, IL-4, IL-6, IL-12, IL-13, IL-17, and IL-22, and regulate B-cell and macrophage activation, macrophage polarization, myofibroblast differentiation, and ECM production [191,194]. B-cells induce autoantibody and cytokine production and affect EC apoptosis and ECM production [191,195]. In addition, B-cell depletion via antibodies against CD20 attenuates fibrosis progression through the suppression of M2 macrophage polarization in mice [196]. Macrophages play a pivotal role in fibrosis progression, and macrophage depletion markedly suppresses fibrosis progression [151]. Macrophages are divided into M1 and M2 macrophages subsets, and M2 macrophages are elevated under conditions of fibrotic disease [197,198]. M2 macrophage polarization is induced by IL-4 and IL-13 [199], and the inhibition of IL-4 and IL-13 signaling by IL-4Rα antibodies suppresses fibrosis progression in mice [151]. The increase in these immune cells induces production of various pro-fibrotic factors and regulates the inflammatory response, vascular homeostasis, myofibroblast differentiation, and ECM production. Abnormality of the innate and adaptive immune system is associated with fibrosis progression.

The Role of the uPA/uPAR System in Inflammation and the Immune System
The uPA/uPAR system regulates cell recruitment, migration, and adhesion, and supports the innate and adaptive immune systems through proteolytic and non-proteolytic mechanisms [200]. Plasmin induced by the uPA/uPAR system has both pro-and antiinflammatory effects and regulates chemotaxis, invasion, phagocytosis, and cytokine production through PAR-1 or Annexin A2 in various cell types, including monocytes, macrophages, and DCs [201][202][203]. In addition, Plg/plasmin regulates macrophage activation, polarization and efferocytosis [204,205]. Furthermore, plasmin can activate complement factors (C3 and C5), factor XII, and MMPs [203]. Plasmin is also associated with the polarization of T cells [206], and mediates the innate and adaptive immune systems.
uPA regulates macrophage chemotaxis, neutrophil activation, and migration through uPAR-dependent or uPAR-independent mechanisms [207]. uPA also mediates the inflammatory response, such as inflammatory cytokine production and suppression of the NF-κB pathway through plasmin activation [34,200]. Furthermore, uPA induces the production of M2 phenotype macrophages [208]. In contrast, uPAR interacts with several receptors, including integrins and LRP, and participates in the initiation of the innate immune response through the induction of cell adhesion and migration [200]. In addition, uPAR regulates toll-like receptor (TLR) 2, 4, and 7 signaling, and affects inflammation (cytokine production, mediation of NF-κB pathway) and immune responses (neutrophil and macrophage activation, macrophage efferocytosis) [38, [209][210][211]. The expression and release of suPAR is induced by inflammation and immune activation [212]. The uPAR-derived ser-arg-ser-arg-tyr (SRSRY) peptide (uPAR88-92 sequence) can interact with FPR1 and is associated with chemokine regulation and monocyte migration [212,213]. The uPA/uPAR system also affects T-cell priming and T-cell effector function [200].
This system has multiple functions and contributes to the inflammatory response and innate and adaptive immune responses.

Conclusions and Therapeutic Perspectives
The uPA/uPAR system regulates proteolysis and intracellular signal transduction through multiple plasmin-dependent and plasmin-independent mechanisms, and mediates vascular homeostasis, the immune system, and ECM homeostasis. This review has presented the latest findings, showing that the uPA/uPAR system plays an important role in fibrosis progression. uPA and uPAR are reportedly associated with several pro-fibrotic events, including vascular endothelial dysfunction (EC injury, apoptosis, defective angiogenesis, EndoMT, and excessive coagulation), immune abnormalities (excessive immune activation, immune cells infiltration, and autoantibodies production), and myofibroblast differentiation. In addition, the importance of uPA and uPAR in fibrosis progression has been proven in several animal models. The control of uPA and uPAR expression, uPA/uPAR binding, and uPAR cleavage may improve vascular endothelial dysfunction, immune abnormalities, myofibroblast and ECM deposition, and fibrosis. The regulation of the uPA/uPAR system through several methods, such as neutralizing antibodies, miRNA, peptides, and protease inhibitors, may be a novel therapeutic approach to managing fibrotic diseases. Further investigations will be required to clarify the role of the uPA/uPAR system in fibrotic diseases.