Systemic sclerosis (SSc) is an autoimmune rheumatic disease of unknown etiology that is characterized by vascular dysfunction and fibrosis of the skin and visceral organs as well as peripheral circulatory disturbance [1]. This process usually occurs over many months and years and can lead to organ dysfunction or death.

In SSc, vascular disorders are observed from early onset to the appearance of late complications and affect various organs, including the lungs, kidneys, heart, and digital arteries, and exacerbate the disease [2]. Microvascular disorders, such as Raynaud’s phenomenon, telangiectasias, and digital ulcers, frequently occur in SSc patients ^[2][3][4][2,3,4]. In contrast, macrovascular disorders, such as those of the coronary arteries, are rarely involved in SSc ^[2][5][6][2,5,6]. In SSc, the vascular dysfunction is caused by vascular and endothelial cell (EC) injury, defective angiogenesis, defective vasculogenesis, endothelial-to-mesenchymal transition (EndoMT), vascular tone alteration, and coagulation abnormalities [7], and is associated with abnormalities in the immune system, such as T-cells, B-cells, mast cells, macrophages infiltration, immune activation, and auto-antibody production, as well as abnormalities in the extracellular matrix (ECM) metabolism, such as myofibroblast differentiation, ECM over-production, and the inhibition of ECM degradation. These abnormalities may influence each other and lead to the development of pulmonary arterial hypertension (PAH) and fibrosis [2]. However, the detailed mechanism underlying the relationship between “fibrosis” and “vascular dysfunction” remains unclear. It is reported that vasculopathy occurs in various mice, as urokinase-type plasminogen activator receptor (uPAR)-deficient mice develop EC apoptosis and severe loss of micro-vessels [8]. Caveolin-1-deficient mice show dilated cardiomyopathy and pulmonary hypertension [9]. Caveolin-1 is associated with the internalization and degradation of transforming growth factor-β (TGF-β) receptors and regulates TGF-β signaling [10]. Fli1-deficient mice show a disorganized dermal vascular network with greatly compromised vessel integrity and increased vessel permeability and impaired vascular homeostasis. Fli1 is associated with the expression of platelet/endothelial cell adhesion molecule (PECAM)-1, platelet derived growth factor (PDGF), and sphingosine-1-phosphate receptors (S1PR) [11]. Fos-related antigen-2 (Fra-2) transgenic mice develop microvascular and proliferative vasculopathy, and pulmonary vascular lesions resembling SSc-associated PAH [12]. However, while these factors may play a critical role in the onset of SSc-associated vascular disorders, the detailed mechanism underlying their involvement is unclear.

2. The Role of Fibrinolytic Regulators in Vascular and EC Injury in SSc

Vascular and EC injury is an early and initiating event in SSc. A number of factors (e.g., infections, cytotoxic T-cells, oxidative stress, auto-antibodies, ischemia-reperfusion) cause persistent EC activation and stimulate the production of various cytokines, EC apoptosis, impairment of cell-cell adhesion, and the activation of complement and coagulant pathways ^[13][98]. In addition, these factors also induce the production of vasodilators, such as nitric oxide (NO), vasoconstrictors, such as endothelin-1 (ET-1), and platelet activation, and lead to the impairment of vascular tone control and vascular and EC damage ^{[2][13][14][15][16]}[2,98,99,100,101].

It is reported that Plg induces EC apoptosis ^[17][102]. Plasmin also damages the endothelial barrier function and EC integrity and induces EC injury ^[18][103]. Plasmin is known to regulate the vascular endothelial function and influence the progression of various cardiovascular diseases through fibrinolysis, the degradation of the ECM, and MMP and TGF-β activation ^[19][20][104,105]. Furthermore, plasmin regulates the fibrin-mediated EC spread and proliferation ^[21][106], MMP-mediated cell adhesion and cell migration ^[22][107], and TGF-β-induced EC apoptosis ^[23][108]. These direct and indirect effects of plasmin may be associated with the maintenance of the endothelial function. Conversely, uPA inhibits EC apoptosis through the induction of X-linked inhibitor of apoptosis protein ^[24][109]. uPAR is involved in the high-molecular-weight kininogen (HKa)-mediated apoptoic effect ^[25][110]. α2AP induces vascular damage, such as the reduction of blood vessels and blood flow in mice, and α2AP neutralization improves vascular damage in SSc model mice ^[26][33]. In addition, α2AP is associated with vascular remodeling and EC apoptosis ^[27][57]. PAI-1 reportedly induces EC apoptosis, but protects against FasL-mediated apoptosis ^[28][29][111,112]. Angiostatin regulates the inhibition of EC proliferation, EC migration, and tube formation, as well as the induction of EC apoptosis ^{[30][31][32][33]}[89,90,91,113]. In SSc, the changes in the expression of the fibrinolytic regulators may regulate the endothelial function and dysfunction.

3. The Role of Fibrinolytic Regulators in Defective Angiogenesis in SSc

In SSc, angiogenesis is incomplete or lacking despite the increased expression of the pro-angiogenic factor VEGF ^[34][114]. VEGF plays a critical role in the maintenance of vascular functions, such as EC growth, activation, proliferation, and migration, through the VEGFR2 signal transduction pathways and also regulates angiogenesis ^[35][115]. The expression of VEGF is elevated in various cells, such as fibroblasts, ECs, and immune cells, but vascular insufficiency manifests in SSc ^[36][37][116,117]. The impairment of VEGF responses may cause vascular dysfunction in SSc, but the detailed mechanisms remain unclear.

Plasmin is known to regulate vascular endothelial functions and influence the progression of various cardiovascular diseases through fibrinolysis, the degradation of matrix proteins, and the activation of growth factors ^[19][104]. In addition, VEGF can be processed by plasmin and thereby released from the ECM ^[38][39][118,119]. α2AP attenuates the VEGF-induced pro-angiogenic effects, such as tube formation and EC proliferation, by blocking the VEGFR2 signal pathway in ECs ^[26][33]. In addition, α2AP is associated with VEGF production in fibroblasts and angiogenesis ^[40][53]. In SSc, fibroblasts are likely to be important effector cells. SSc fibroblasts inhibit angiogenesis and induce vascular dysfunction ^[1][26][41][1,33,120]. The blocking of α2AP markedly improves the SSc dermal fibroblast-induced vascular dysfunction, indicating that SSc fibroblast-derived α2AP affects vascular dysfunction in the disease ^[26][33]. An increased α2AP expression in SSc may cause impairment of the VEGF response and lead to vascular dysfunction. uPA and uPAR play important roles in angiogenesis and modulate the VEGF signaling ^[42][43][121,122]. uPA and uPAR are associated with the impairment of angiogenesis in SSc, and the SSc EC-conditioned medium attenuates uPA-dependent EC proliferation and invasion. In addition, the cleavage of uPAR by the overproduction of MMP-12 in SSc inhibits angiogenesis ^[41][44][120,123]. uPAR can interact with integrins, which mediate actin assembly in ECs and are associated with angiogenesis and vascular alterations in SSc ^[45][46][47][124,125,126]. uPAR also regulates VSMC proliferation and migration ^[48][49][127,128]. PAI-1 inhibits the binding of VEGFR-2 to β3 integrin as well as VEGF signaling ^[50][129]. In addition, PAI-1 binds to uPA and uPAR to exert anti-angiogenic effects ^[51][130]. tPA induces VEGF production through the ERK and p38 pathways in ECs ^[52][131].

Angiopoietins regulate vascular homeostasis through the Tie2 receptor ^[53][54][55][132,133,134]. Angiopoietin-1 (Ang-1) mediates vascular remodeling and stabilization, while angiopoietin-2 (Ang-2) functions as a Tie2 agonist or antagonist and is associated with angiogenesis and vascular permeability ^[54][56][133,135]. Ang-1 is decreased while Ang-2 is increased in the sera of patients with SSc and the differential expression of Ang-1/Ang-2 may be associated with the progression of SSc ^[57][136]. tPA regulates Ang-2 production ^[58][137], so an increase in tPA may induce an increase in Ang-2. In addition, α2AP inhibits the Ang-1-induced EC sprouting ^[59][138], and the suppression of uPA and uPAR inhibits Tie2 activation and attenuates angiogenesis ^[60][139]. Ang-1 or Tie2 can interact with integrins ^[61][62][140,141]. α2AP or uPA/uPAR-mediated Tie2 activation may be associated with the binding of integrins.

Angiostatin is known to be an anti-angiogenic factor that regulates EC proliferation, EC migration, EC apoptosis, and VEGF expression while inhibiting angiogenesis ^{[30][31][32][63]}[89,90,91,92]. Angiostatin is generated by elastase ^[64][84]. MMP-12 is a macrophage elastase, and MMP-12 is elevated in SSc ^[41][120]. This increase in the MMP-12 expression may cause angiostatin overproduction, thereby leading to defective angiogenesis.

4. The Role of Fibrinolytic Regulators in Coagulation Abnormalities in SSc

Microvascular thrombosis and fibrin deposition were observed in patients with SSc, and an imbalance in coagulation and fibrinolysis causes vascular damage ^[2][14][65][2,99,168]. The levels of von Willebrand factor (vWF), fibrinogen, ET-1, sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) are elevated in SSc ^[2][14][2,99]. In addition, a specific nonintegrin receptor for type I collagen was found to be elevated in platelets obtained from SSc patients, and an increased responsiveness of SSc platelets to 5-hydroxytryptamine (5HT), adrenaline, ADP, and collagen were reported ^[66][67][169,170]. Those increases may cause the activation of platelets and hypercoagulation. Furthermore, plasmin induces platelet activation, platelet aggregation, and platelet release reaction through PAR ^[68][69][70][171,172,173]. Plasmin also enhances their sensitivity to ADP ^[70][173]. In SSc, increases in the levels of uPA and tPA may promote plasmin generation and the activation of platelets, which synthesize and release α2AP and PAI-1 ^[71][72][174,175].

The expression of α2AP and PAI-1 ^[73][74][24,31] and uPAR cleavage by MMP-12 overexpression ^[41][120] is elevated in SSc. Furthermore, α2AP can be crosslinked to the fibrin surface by activated FXIIIa ^[75][63], and PAI-1 binds to fibrin through Vn ^[76][176]. The inactivation of plasmin by increases in the expression of α2AP and PAI-1 may cause the impairment of fibrinolysis. In addition, Barrett et al. suggest that the angiostatin generation induced by elastase-degraded Plg may underlie the fibrinolytic shutdown ^[77][87]. These changes in fibrinolytic regulators may cause the impairment of fibrinolysis and lead to the deposition of fibrin and coagulation abnormalities characteristic of SSc.

5. The Role of Fibrinolytic Regulators in Vascular Tone Alteration in SSc

In SSc, it has been reported that the eNOS expression and NO release are decreased, and the impairment of NO response attenuates vasodilation ^[14][99]. Conversely, vasoconstrictors, such as ET-1, are elevated in SSc and cause abnormal vasoconstriction ^[14][99]. These changes in the vascular tone in SSc may lead to vascular damage. tPA, PAI-1, and plasmin inhibitor have been reported to modulate vasodilation and vasoconstriction and regulate the vascular tone ^[78][79][177,178]. In addition, PAI-1 deficiency prevents hypertension in response to long-term NOS inhibition ^[80][179], and uPA promotes the LRP-mediated eNOS activation ^[81][180]. Furthermore, angiostatin inhibits the VEGF-induced NO production and is involved in vasodilation ^[82][83][181,182]. The fibrinolytic system may be involved in the vascular tone alterations observed in SSc.