Systemic sclerosis (SSc; scleroderma) is a systemic connective tissue disease that affects the capillaries and small arteries, resulting in reduced blood flow, tissue ischemia, and defective wound healing (1). The spectrum of vascular abnormalities in SSc extends from Raynaud's phenomenon, with reduced blood flow to the skin, finger ulcerations, and the potential for gangrene (2), progressive pulmonary hypertension, and coronary microvascular disease that can result in myocardial ischemia, contraction band necrosis, and fibrosis (3), to abnormal tissue healing leading to scarring and sclerosis (3). SSc vascular disease also affects other organs, including the kidneys and the gastrointestinal tract. Although the pathogenesis of SSc is not completely understood, the 3 main components of the disease process include autoimmunity, tissue fibrosis, and aberrant microvascular function.
Vascular homeostasis requires both proangiogenic and antiangiogenic factors. The proangiogenic factors are secreted molecules that promote endothelial cell proliferation, migration, and tubulogenesis (4, 5). Cleavage products of several extracellular proteins, including matrix proteins such as type XVIII collagen and type IV collagen, and hemostatic proteins such as plasminogen, among others, have been shown to possess antiangiogenic activity (6, 7). The activity of these factors as well as other proteins controlling angiogenesis is tightly regulated in quiescent tissue. Its alteration under pathologic conditions may result in abnormal growth of the vasculature or defective repair processes. Earlier reports have suggested the presence of antiendothelial activity in SSc, but its nature has not been defined (8–10).
Plasminogen plays a complicated role in the regulation of vascular homeostasis because of its ability to serve as a precursor of both the protease plasmin that possesses proangiogenic activity (11) as well as the angiogenesis inhibitor angiostatin (12). Plasminogen cleavage by plasminogen activators at residues Arg561–Val562 produces plasmin, which has both proteolytic and fibrinolytic activities (13). The proteolytic activity controls activation of several important angiogenic modulators such as transforming growth factor β and matrix metalloproteinase (MMP) (14, 15). Plasmin also activates and releases growth factors such as vascular endothelial growth factor and fibroblast growth factor, thereby promoting angiogenesis.
Plasminogen can also be cleaved by several proteases, including urokinase plasminogen activator (uPA), tissue plasminogen activator (16), MMP-2, MMP-3, MMP-7, MMP-9, and MMP-12 (16–19), and plasmin reductase (20, 21) within its kringle domains, producing angiostatin fragments of various sizes that possess antiangiogenic activity. Thus, alterations in plasminogen processing can have a profound effect on angiogenic homeostasis.
The present study was designed to explore the hypothesis that patients with SSc demonstrate excess antiangiogenic activity that can account for the vascular abnormalities seen in this disease. To this end, plasma samples obtained from patients with SSc and from age- and sex-matched control patients were evaluated for antiangiogenic activity. Plasma from patients with SSc, but not that from control patients, markedly inhibited the angiogenic properties of normal endothelial cells. The SSc plasma contained reduced amounts of proangiogenic plasmin and increased levels of antiangiogenic angiostatin, which was produced by proteases in T cell granule content cleaving plasminogen at kringles 3 and 4. Thus, we observed that alterations in plasminogen processing may play an important role in the pathophysiology of SSc.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we showed that exposure of normal endothelial cells to plasma from patients with SSc results in a significant inhibition of 2 proangiogenic functions: endothelial cell migration and the ability to form vascular structures in 3-D collagen gel. We also demonstrated the presence of 3 abnormalities of the plasminogen system in SSc patients: reduced plasmin activity, greater amounts of glutamic acid–plasminogen versus lysine–plasminogen, and increased amounts of plasminogen and plasmin cleavage products containing angiostatin K1–4.5 and K1–3 fragments. Proteases released from T cell granules appear to be responsible for angiostatin production and reducing plasmin levels in patients with SSc. Taken together, these results suggest that SSc is associated with a systemic state that results in inhibition of angiogenesis, and that these defects might be related, in part, to abnormalities of the plasminogen system and production of angiostatin due to plasma release of T cell granules.
Abnormal angiogenesis in patients with SSc has been suggested as a cause of some of the key features of the clinical syndrome, including abnormal wound healing (34) and progressive pulmonary hypertension. Several studies have demonstrated angiogenesis-related abnormalities, including decreased monocyte-mediated angiogenesis (35) and the presence of proangiogenic and antiangiogenic factors in SSc serum (36), including increased levels of circulating endostatin (37, 38). At the same time, SSc serum was reported to enhance the angiogenic capability of circulating mononuclear cells (39). Finally, a recent gene expression study comparing transcriptomes of microvascular endothelial cells from normal control subjects and patients with SSc demonstrated overexpression of proangiogenic transcripts and both up-regulation of suppressor genes and down-regulation of activator genes regulating cell migration in SSc endothelial cells (40). Thus, to date, there is no clear understanding of the prevalence and significance of angiogenic abnormalities in SSc (41).
In this study, we set out to examine the function of the plasminogen system in SSc. Plasminogen is the source of both plasmin, a serine protease with marked proangiogenic properties, and angiostatin, an antiangiogenic peptide. The demonstration of strong antiangiogenic activity of SSc serum that was maintained in 1:1 ratio mixing studies suggested the presence of a circulating inhibitor and prompted us to examine in detail the components of the plasminogen system. We observed both a significant decrease in plasmin activity as well as a significant increase in angiostatin levels compared with those in control patients, thereby tipping the balance toward antiangiogenesis.
To investigate factors responsible for decreased plasmin activity, we first evaluated the amount of plasminogen and the presence of its inhibitor, PAI-1. However, we detected no differences in the level of either plasminogen or PAI-1 between the groups, suggesting that neither plasminogen nor PAI-1 could account for reduced plasmin activity. It should be noted that some studies have shown elevated PAI-1 levels in fibroblasts from patients with SSc (42–45), while other studies have not (46–48). It is not clear why results differ in this regard, other than due to relatively small sample sizes in all studies and the high heterogeneity of the SSc patient population.
We then turned our attention to changes in plasminogen structure that could account for reduced plasmin generation. Plasminogen can be present in either the glutamic acid–plasminogen form (which contains residues 1–77) or the lysine–plasminogen form (in which residues 1–77 have been removed). Glutamic acid–plasminogen residues 1–77 maintain it in a compact conformation through its intramolecular binding interactions with lysine residues in kringles 4 and 5 (49), thereby reducing activation of plasminogen into plasmin (50). An examination of glutamic acid–plasminogen versus lysine–plasminogen levels in SSc and control plasma showed an abundance of full-length glutamic acid–plasminogen in SSc plasma. Thus, this shift in plasma plasminogen content from the lysine to the glutamic acid form can be one factor explaining low plasmin activity in SSc plasma.
We then evaluated the presence of angiostatin, a plasminogen-derived fragment, in plasma from patients with SSc. Individual plasminogen kringles and various subsets of kringle domains have angiostatin activity, and many are more potent angiogenesis inhibitors than the 38-kd fragment containing K1–4, which was originally identified as angiostatin (51–53). Thus, the K1–3, K1–5, and K1–4.5 (kringles 1–4 plus 85% of kringle 5) fragments exhibit more antiangiogenic activity than K1–4 (12, 54, 55). Remarkably, we detected the presence of several “angiostatins” in the SSc patient plasma, including the presence of a 25-kd form (lysine K1–3). Furthermore, patient plasma containing angiostatin was significantly more inhibitory to normal endothelial cells, suggesting the functional role of angiostatin. This was further confirmed by demonstrating a similar extent of inhibition of angiogenic endothelial cell function by the addition of purified angiostatin in a concentration similar to that observed in patients with SSc.
There are numerous reports of enzymes that cleave plasminogen at the kringle domains to produce angiostatin containing either K1–3, K1–4, and K1–4.5 or combinations of the kringle subsets. Among those reported are various MMPs (17–19) and plasmin reductase (20, 21). Granzyme B, a serine protease found in T cell granules, is strikingly associated with cleavage of autoantigens targeted by autoantibodies in patients with SSc (33, 56). In agreement with this hypothesis, we demonstrated that both granzyme B and the full granule content could indeed cleave plasminogen into various-sized fragments containing angiostatin K1–3. Proteolysis appeared to be a 2-step process in which fragments K1–4.5 underwent further proteolytic processing. Interestingly, there was a highly significant correlation between the level of granzyme B and angiostatin in SSc plasma.
Although we detected a significant correlation between plasma granzyme B and K1–3 fragment levels, the pattern of granzyme B cleavage of plasminogen implied that a second protease is required to achieve the angiostatin K1–3 fragment at 25 kd seen in plasma from patients with SSc. Furthermore, the data clearly showed that granule content, but not granzyme B, was required to achieve cleavage into a 25-kd angiostatin fragment. Thus, we conclude that both granzyme B and another protease derived from T cell granule content is necessary for angiostatin generation in patients with SSc.
It remains unclear why plasmin levels are reduced in SSc plasma. We considered that granzyme B and/or another granule content protease may cleave plasmin to regulate plasmin conversion of glutamic acid–plasminogen into lysine–plasminogen. When incubated with plasmin, both granzyme B and granule content cleaved the intact plasmin into K1–3 fragments ranging from 29 kd to 54 kd. Granzyme B cleavage of plasmin also contained a 25-kd angiostatin fragment. The elevated levels of granzyme B, and by implication, granule content in the SSc plasma, explain in part the reduction in plasmin activity observed in patients with SSc.
Thus, patients with SSc display 2 principal abnormalities of the plasminogen system: a reduction in conversion of glutamic acid–plasminogen to lysine–plasminogen and production of 25-kd angiostatin. We propose that the primary abnormality is the release of T cell granule content that leads to cleavage of both lysine–plasminogen and plasmin to angiostatin. In addition to an increase in 25-kd angiostatin production, the presence of the T cell granule content proteases in plasma, including granzyme B, leads to a reduction in the plasmin level due to its conversion to angiostatin, which leads to reduced conversion of glutamic acid–plasminogen to lysine–plasminogen (Figure 3D). Thus, the release of T cell granule content is likely the primary event that accounts for all of the observations in this study: increased angiostatic activity in SSc patient plasma, increased presence of the 25-kd angiostatin, decreased conversion of glutamic acid–plasminogen to lysine–plasminogen, and reduced plasmin activity.
The presence of an increased amount of angiostatin, therefore, is likely responsible for impaired angiogenesis in some but not all patients with SSc. Furthermore, it is interesting to speculate that in addition to contributing to the antiangiogenic activity of SSc plasma, the presence of increased amounts of angiostatin may be a marker of increased matrix degradation, which is expected in patients with SSc. Some such degradation products have been shown to possess antiangiogenic activity, including the type XV/XVIII collagen breakdown product endostatin (6, 57) and the type IV collagen breakdown product tumstatin (58), among others (7).
In summary, we demonstrated that SSc is associated with 2 significant abnormalities of the angiogenic cascade: decreased presence and activity of proangiogenic plasmin, and increased production of antiangiogenic angiostatin. Both abnormalities are generated by the proteolytic activity of granzyme B and a second protease found in granule content. The data suggest a novel mechanism contributing to vascular defects in SSc that links immune-mediated proteolysis and altered angiogenesis. This insight may provide the opportunity to investigate novel therapeutic options.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Simons had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Mulligan-Kehoe, Drinane, Rosen, Wigley, Simons.
Acquisition of data. Drinane, Mollmark, Casciola-Rosen, Hummers, Hall, Wigley.
Analysis and interpretation of data. Mulligan-Kehoe, Casciola-Rosen, Rosen, Wigley, Simons.
Manuscript preparation. Mulligan-Kehoe, Hummers, Rosen, Wigley, Simons.
Statistical analysis. Mulligan-Kehoe, Hummers, Wigley, Simons.