In the present study were found that concentrations of NO metabolites were not increased in patients with both SSc and PAH, although plasma ET-1 levels were markedly elevated. Our previous report [21
] indicates that serum levels of NO metabolites were significantly higher in patients with SSc than in healthy control individuals, especially in patients with a diffuse cutaneous type, active fibrosing alveolitis, or a short duration since onset. However, the population considered in that study did not include patients with PAH, which could explain why the present findings are inconsistent with those of the previous report. Although a number of reports have been published concerning concentrations of ET-1 or NO in the circulation of patients with SSc [8
], this report is the first to describe an imbalance in the NO/ET-1 ratio in patients with PAH.
Over the past decade abnormalities in NO synthesis have been proposed as being important in the pathogenesis and development of pulmonary hypertension, especially primary pulmonary hypertension (PPH). Initially, immunohistochemical studies showed that pulmonary hypertension was associated with diminished expression of NOS-3 [37
]. However, other studies found increase in expression of NOS-3 in patients with pulmonary hypertension and in animal models of pulmonary hypertension [38
]. Despite these contradictory findings, it has been reported that NO levels in blood and the lungs were precisely decreased in patients with PPH and collagen disease related PAH [8
]. Furthermore, it was determined that NOS-dependent endogenous NO synthesis was decreased in patients with PPH, which suggests that NOS activity may be diminished in patients with PPH [40
]. Lung inflammation leading to increased levels of cytokines and oxidants may contribute to the development of both PPH and SSc-related PAH [41
]. In the presence of increased levels of inflammatory mediators, NOS activity may be dependent on production of NOS-2, which is distinct from NOS-3 (the endothelial form of NOS) because NOS-2 is inducible by inflammatory mediators, and induced levels are much greater than levels of constitutive NOS-3 production. Peripheral mononuclear cells and lesional fibroblasts are capable of aberrant production of inflammatory cytokines in patients with SSc [42
]. These cytokines may be involved not only in ET-1 synthesis by endothelial cells and fibroblasts but also in induction of NOS-2. Also, excessive production of ET-1 can mediate NOS-2 production through ET receptor B [45
]. Although evidence based on those biological properties may promote speculation that levels of ET-1 correlate with levels of NO in the circulation, NO metabolite levels were within normal range in patients with both SSc and PAH patients whose serum contained much ET-1. We hypothesize that this discrepancy may be explained by reduced NOS-2 production resulting from polymorphisms in the NOS2
As a result of sequencing the promoter region of the NOS2
gene from -100 to -1,335, we were able to confirm the presence of two SNPs, consistent with previous reports [46
]. In the present study, allele A at -277 SNP, allele G at -1,026 SNP and shorter forms of the CCTTT repeat were associated with susceptibility to PAH combined with SSc. The number of CCTTT repeats was previously reported to influence transcription of the NOS2
]. However, studies of variable numbers of tandem repeat both in vitro
and in vivo
have yielded conflicting results [48
]. To confirm whether those polymorphisms affect transcription of the NOS2
gene in fibroblasts, we constructed a series of luciferase reporter vectors cloned by various numbers of CCTTT combined with the promoter region of the NOS2
gene from +58 to -1,557, which included two kinds of haplotype.
Transcriptional activity was lowest in the NOS2 gene containing the six repeats of CCTTT and haplotype GA, which suggests that transcription of the NOS2 gene might be little induced by interleukin-1β in patients with SSc-related PAH.
Irrespective of whether patients with SSc had PAH, CCTTT repeat length was well correlated with NO/ET-1 ratio. With regard to the relationship between CCTTT repeat length and serum NO levels, we found no significant difference among SSc patients without PAH, although there were significant differences among all SSc patients and among patients with both SSc and PAH (data not shown). In the setting of aberrant production of ET-1 or cytokines, NO synthesis via NOS-2 induction may be dependent on NOS2 gene polymorphisms. In healthy control individuals, who had no vascular damage, inflammation, or autoimmune disorders, there was no association between CCTTT repeat length and either serum NO levels or NO/ET-1 ratios (data not shown). Because NOS-2 induction is well controlled by ET-1 and cytokines, distinct from NOS-3, which is constitutively produced, it has been suggested that the CCTTT repeat length is more significantly correlated with NO/ET-1 ratios than with serum NO levels. Our observations support the concept that the NOS2 gene polymorphism is a crucial factor in NO synthesis under conditions of vascular damage and chronic inflammation, as well as PAH.
It is not possible to determine whether SSc patients without PAH will suffer this complication in the future, and this is a limitation of the present study. The patients enrolled in the study are from a prospective cohort at our institution, and they have been observed for clinical complications, including PAH, in the follow-up clinic. None of the 58 patients with SSc but not PAH has yet been diagnosed with PAH (mean duration of observation: 45 months).