Systemic sclerosis (SSc) is an autoimmune disorder of the connective tissue, characterized by widespread vascular lesions and fibrosis. Raynaud's phenomenon, increased thickness of the vascular wall, devascularization, and thickness of the basement membrane are typical features of SSc. The digital arteries of patients with SSc exhibit marked intimal proliferation, resulting in severe narrowing and occlusion of arterial lumen and limited vasodilative responses to vasodilator therapies (1).
Thus far, the contributions of many molecules to the pathogenesis and diagnosis of SSc have been reported (2–9). Investigation of these known molecules is of great value for the understanding of SSc pathogenesis; however, it would also be important to identify novel SSc-related molecules by hypothesis-free comprehensive surveillance. In this regard, genomic surveillance using single-nucleotide polymorphisms, DNA arrays for messenger RNA expression, and proteomic surveillance have been used recently (10). These techniques can detect various molecules that may be involved in the pathogenesis of SSc or that may be useful for diagnosis. However, the targets of surveillance have been limited to genes or relatively large proteins. Peptides with low molecular weights (smaller than ∼3 kd) generally cannot be investigated by these techniques.
Peptides with such low molecular weights often play a central role in biologic and pathologic processes. Typical of these peptides would be bradykinin, which is a peptide that is only 9 amino acid residues in length and produced from kininogen by specific proteolysis. Another example would be substance P, in a neuropeptide of 11 amino acid residues. Peptides with such low molecular weights are estimated to be produced in large amounts by proteolysis of large proteins, which thereby generates various bioactive and/or diagnostically useful short peptides in addition to the known peptides in the body. However, there are only a few ways to survey such peptides efficiently. Quite recently, mass spectrometry methods that directly detect and identify peptides with low molecular weights and with low concentrations (referred to as peptidomics analysis) have been developed. This offers a promising approach to the discovery of novel short peptides that could be useful in the diagnosis and treatment of diseases (11–13).
In the present study, we aimed to identify low molecular weight peptides in the serum of patients with SSc in order to understand the role of these peptides in disease pathogenesis, which could lead to better diagnosis and treatment of SSc. By combining a microamount peptide-separating method with magnetic beads and matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry, we were able to comprehensively detect short peptides in the sera of patients with SSc, those with non-SSc rheumatic diseases, and healthy donors. In comparing the results between each group of samples, we found that the sera of patients with SSc carried complement C3f-des-arginine (DRC3f) and its degraded smaller fragments at very high levels and with high frequency, compared with the sera of patients with non-SSc rheumatic diseases or healthy donors. Furthermore, we demonstrated that these peptides promoted proliferation of human microvascular endothelial cells (HMVECs) in vitro. The results of this study will open a new field of investigation into the pathophysiologic processes of SSc.
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This study is the first to comprehensively survey disease-related short peptides in patients with SSc, since there have been no efficient ways to approach this in a comprehensive manner thus far. By combining purification of short peptides with C18-bound magnetic beads and peptidomics analysis by a mass spectrometry–based technique, we successfully detected and identified a series of short peptides that exist predominantly in the sera of patients with SSc. Our findings were as follows. 1) The short peptides detected predominantly in patients with SSc as compared with those with SLE, RA, or OA and healthy donors were identified as DRC3f, a fragment produced by the inactivation process of C3b, and its degraded derivatives. 2) The relative concentration of DRC3f was related to several clinical features and parameters of vascular involvement and disease activity in SSc. 3) Functionally, synthesized peptides of DRC3f and C3f were demonstrated to enhance proliferation of HMVECs and increase HMVEC production of TGFβ1.
The complement system has been shown to be involved in the pathogenesis of SSc. Genetically, the null allele of C4A*Q0 was reported to have a strong association with SSc (23, 24). Increased serum concentrations of C1q, C2, C5, C6, C7, C9, and factor B and a decreased concentration of C4 have been observed in patients with SSc (25). Increased concentrations of C3d and C4d and increased ratios of C3d:C3 and C4d:C4 were reported to have a positive relationship with the severity of SSc (26). Another study also showed that an increased C3d level and markedly decreased complement level were dependent on prevention of immune precipitation in patients with SSc (27). In that study, immune precipitation was suppressed significantly in patients who carried the C4A*Q0 allotype (27). More recently, the activated complement complex C5b-9 and the C5a receptor were detected in the microvasculature of SSc patients, both in the early and in the late stages of the disease (28, 29).
These findings suggest that both abnormal complement activation and complement heredity are involved in the pathogenesis of SSc. Activated complement may impair the membranes of endothelial cells directly by C5b-9, resulting in endothelial cell death and increased permeability of the endothelium. Furthermore, fragments released from complement activation, such as C3a and C5a, are strong chemotaxins, which attract leukocytes into sites of inflammation (30). Other fragments produced in the process of activation/inactivation of complement, such as C3b, iC3b, and C3d, may have biologic functions. However, it remains uncertain whether these degraded fragments of complement play a pathogenic role in SSc.
Complement C3 is a key molecule in the formation of C5 convertase. Complement activation includes 3 main pathways of the classical pathway, mannose-binding lectin pathway, and alternative pathway. In healthy conditions, only low-level spontaneous C3 activation occurs, via the alternative pathway. C3b, produced by C3 activation, is degraded to form inactive iC3b with the help of complement receptor 1 (CR1) or factor H, while, simultaneously, C3f of a 17-amino-acid peptide is cleaved from C3b. C3f is further degraded to form DRC3f via carboxypeptidase N, which leads to release of the C-terminal arginine, the main form of C3f derivatives (20). Thus, DRC3f might also be a marker of complement activation. In fact, our results showed that the dramatic increase in serum levels of DRC3f in SSc patients had a negative correlation with the serum C3 and C4 levels.
However, it remains unclear why the DRC3f levels were much higher in patients with SSc than in patients with SLE, the classic immune complex disease. The increased basic levels of complement components, including C3, in those with SSc might have partially contributed to elevations in the DRC3f level (25), whereas the reduced levels of complement regulators, including CR1, and quick depletion of the complement components (31) might have contributed to the lower level of DRC3f in those with SLE. It would be of value to investigate the activity of complement regulatory proteins, including CR1, factor H, and factor I, in SSc.
We found that the DRC3f level had a significant correlation with the level of IgA. In a previous study, IgA-containing immune deposits were detected at vascular sites in patients with SSc (32). Moreover, IgA in immune aggregates deposited at vascular sites was demonstrated to trigger activation of the complement cascade through the alternative pathway (33, 34). Both of these features of IgA may play important pathologic roles in SSc. Functionally, both C3f and DRC3f have been demonstrated to enhance vascular permeability and induce smooth muscle contraction as a weak spasmogen (20). The hexapeptide HWESAS, a sequence encompassed within C3f, has recently been found to potentiate the sulfation and mitogenic activities of the IGFs (22). Based on these findings in addition to our own results, we hypothesized that DRC3f could play a role in the pathogenesis of SSc.
To test this point, we synthesized both C3f peptides and DRC3f peptides and observed the functions of both peptides in vitro. Our results showed that the synthesized C3f and DRC3f promoted proliferation of HMVECs independent of any role of IGF. In addition, we showed that both the DRC3f-containing serum samples and the filtered DRC3f-containing serum samples (containing only molecules with molecular weights of lower than ∼3 kd) enhanced the proliferation of HMVECs (Figure 4). These results suggest that DRC3f is one of the low molecular weight growth factors in sera (22, 35). However, morphologic abnormalities in the vessels, defective angiogenesis, and apoptosis instead of proliferation of endothelial cells are common features of the microvasculature in SSc. This appears to be consistent with the increased DRC3f levels observed in the present study.
In contrast with the findings in skin HMVECs, lung HMVECs have been demonstrated to proliferate in bleomycin-induced pulmonary fibrosis in rats (36), in SSc patients with fibrosing alveolitis (37, 38), and in SSc patients with pulmonary hypertension (39). We found that HMVECs from a lung cell line showed a higher frequency of proliferative responses to DRC3f and C3f than did HMVECs from a skin cell line. Moreover, the DRC3f level was associated with ILDs in patients with SSc. Thus, DRC3f may play a role in the pathogenesis of SSc in association with ILDs and/or pulmonary hypertension.
It has been demonstrated that growth factors, including TGFβ, are responsible for proliferation of dermal fibroblasts and small artery smooth muscle cells, and for excessive production of extracellular matrix components such as types I, III, V, and VII collagens and fibronectin (40). We therefore also investigated the influence of DRC3f and C3f on the production of TGFβ1, VEGF, and EGF, using the same cell lines as above. Both DRC3f and C3f markedly enhanced production of TGFβ1 by the HMVECs of a skin cell line, but not by the lung HMVECs or a human dermal fibroblast cell line. None of the 3 cell lines were found to produce detectable amounts of VEGF and EGF after stimulation with either DRC3f or C3f. Thus, in our study, DRC3f and C3f failed to show a growth factor–like potential, at least with regard to the expression of TGFβ1, VEGF, and EGF, even though a part of this potential could be attributed to expression of TGFβ1 in one of the tested cell lines. Alternatively, DRC3f and C3f may have a direct influence on HMVECs. To resolve this issue, we are currently investigating target molecules of DRC3f and C3f, using a comprehensive proteomics approach.
With regard to clinical features, the DRC3f level was associated with higher levels of antinuclear antibodies, rheumatoid factor, IgG, IgA, and CRP, lower levels of C3, C4, and CH50, an accelerated ESR, and presence of anti–topoisomerase I and anti-RNP antibodies. These findings indicate that DRC3f could be linked to pathologic immune reactions in SSc. In addition, the DRC3f level was associated with ILDs, digital pitting scars, sicca symptoms, and esophageal involvement. DRC3f therefore appears to be linked to the activity and severity of SSc, including vascular involvement. However, the DRC3f level did not correlate significantly with the modified Rodnan total skin thickness score, which is a measure of the main feature of SSc.
In addition, the level of DRC3f was associated with long disease duration, but the activity of SSc is often higher in the early stages of the disease. It is also puzzling that DRC3f levels were associated with the presence of ILDs but not with KL-6 and surfactant protein D. Therefore, we must conclude, based on the available evidence, that the linkage of DRC3f to the activity and severity of SSc might be only partial. To resolve this issue, future investigations into the mechanisms by which DRC3f contributes to the clinical features of SSc are needed.
In summary, DRC3f and its smaller derivatives, produced in the process of inactivation of C3b, were detected predominantly in the sera of patients with SSc in association with disease activity and vascular involvement. We further demonstrated DRC3f to be a low molecular weight growth factor that enhanced proliferation of HMVECs and, in some cells, enhanced TGFβ1 production. These results suggest that the activation of complement and the effects of DRC3f each play a crucial role in the pathogenesis of SSc. Future studies should focus on the functions of DRC3f in SSc sera, since this would provide us with valuable information regarding the pathophysiologic mechanisms of the disease.