Curator: Aviral Vatsa, PhD, MBBS
Systemic sclerosis (SSc) is a type of autoimmune disease when the body’s immune system attacks and destroys body’s healthy tissue. It is characterised by lesions in the vessels and accumulation of collagen in the tissues. Although the pathogenesis of this disease is not clear, but one of the suggestions is that the endothelium fails to produce NO upon cold stimulation. Physiologically, NO acts as a vasodilator and its deficiency has been implicated in diseases such as hypertension and atherosclerosis.
In the body NO is generated when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS.
The inducible type 2 NOS (iNOS) may act as an immunoregulator. Several reports have provided evidence for the existence of a NO pathway in human mononuclear cells.
It is not well established if NO production increases or decreases in SSc patients. In one study by Allanore et al, NO production was shown to be reduced in plasma and PBMC supernatants, and iNOS synthesis in PBMCs.
The authors of this study investigated NO metabolites in plasma and PBMC supernatants, and iNOS synthesis in PBMC to see if the level of NO production by peripheral blood mononuclear cells (PBMC) was low in SSc, as this might contribute to the vasodilatory abnormalities observed in this disease.
Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica.The NO metabolites nitrite and nitrates were determined by flurometeric and spectrometeric assays respectively. iNOS expression was determined by using monoclonal anti‐NOS2 antibody and FACS analyses.
The data suggested a decrease in plasma NO concentration and iNOS production in PBMC in patients with systemic sclerosis as compared with patients with rheumatoid arthritis and sciatica. Subgroup analysis showed no difference between limited and diffuse SSc forms. Total plasma nitrite concentrations in five healthy volunteers were similar to those in patients with sciatica, which is consistent with this group being an appropriate control group.
Thus the authors suggest that low NO production in Ssc patients might be involved in the tendency towards vasospam.
However in other studies authors have shown an increase in NO production in SSc patients. Takagi et al set out to investigate this discrepancy in NO production in SSc patients. They sought to determine whether increased NO levels are associated with various clinical subsets of SSc patients, and to assess the contribution of fibroblasts in skin lesions to NO synthesis.
In this study Takagi et al measured the levels of serum NO metabolites in SSc patients and determined the contribution of the excessive production of NO synthase (NOS)-2 by skin fibroblasts to NO synthesis. Serum NO levels of 45 patients with SSc were significantly higher than those of 20 healthy volunteers. In addition, some clinical features of SSc (the extent of skin fibrosis, short disease duration, and the complication of active fibrosing alveolitis) were all correlated positively with the levels of NO metabolites in SSc patients. RT PCR was used to determine NOS-2 mRNA expression levels in cultured fibroblasts derived from SSc patients.
The authors showed that serum NO levels were significantly elevated in patients with SSc as compared to healthy normal controls. They also demonstrated that NOS-2 was produced spontaneously by cultured SSc fibroblasts, suggesting that increased serum NO levels might reflect in part the elevated expression of NOS-2 by fibroblasts derived from SSc patients.
The discrepancy in NO production could be explained by disease stage, severity of tissue fibrosis and various circumstances of endothelial damage. Takagi et al found increased NO production in early stages of SSc with tissue fibrosis and not in later stages of the disease. Hence they suggest that NO levels may be a sensitive marker of the early stages of the development of severe tissue fibrosis in SSc patients, although a longitudinal and prospective study is needed to confirm this.
NO is known to have dual functions in the body, both beneficial and cyototoxic. Generally it depends upon the concentration and the duration of NO production. Similarily in SSC, the dual functions of NO seem to be both beneficial (as a vasodilator) and harmful (as a cytotoxic effector) in regard to the clinical manifestations of SSc. One of the limitations of these studies is that they did not investigate the absolute concentrations of NO production but only its metaoblites were measured. This might be due the fact that NO is a short-lived molecule (half-life < 5 s) and it is challenging to quantify NO production at single cell level. Such techniques (e.g.DAR 4M AM flurophore) have been developed but are challenging to apply to various experimental set ups. DAR 4M AM has been used to quantify NO production online in single cells. In SSc determination of absolute NO concentrations at cellular or tissue level at various stages of the disease process will go a long way in solving the discrepancy of NO production in SSc patients.
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