MABC1104-100UG Sigma-AldrichAnti-Kallistatin (PI-4) Antibody, clone G4C10

Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor forkhead box O 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahl salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, H(2)O(2) dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO1 expression with small-interfering RNA. H(2)O(2) rapidly induced FOXO1 nuclear translocation, but the effect was blocked by c-Jun NH(2)-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed H(2)O(2)'s effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells.

Previous studies demonstrated suppression of rat ankle arthritis by local injection of kallistatin gene, a negative regulator of angiogenesis. We analyzed circulating levels, synovial concentrations, and tissue localizations of kallistatin in patients with rheumatoid arthritis (RA).Paired plasma and joint fluid samples were simultaneously obtained from 24 patients with RA and 14 with osteoarthritis (OA). Synovial tissues from 5 patients with RA and 5 with OA were obtained during surgery. Fibroblast-like synoviocytes (FLS) and mononuclear cells (MNC) were prepared. ELISA was used to measure kallistatin levels of plasma, joint fluid, cell lysate, and synovium homogenate extract. Synovial tissues were subjected to Western blot and immunohistochemical staining. In addition, the tissue kallikrein (TK) levels of plasma and joint fluid samples were also measured by the ELISA.Circulating and synovial levels of kallistatin and TK were elevated in patients with RA. The immunohistochemical assay exhibited stainings of kallistatin on both infiltrating MNC and FLS. Intracellular kallistatin levels were significantly elevated in MNC and FLS from patients with RA.Elevated kallistatin levels were demonstrated in patients with RA, particularly in synovial tissues, FLS, and MNC. This report is the first to demonstrate upregulation of kallistatin expression in rheumatoid joints.

Kallistatin, a human serine proteinase inhibitor, is a newly identified tissue kallikrein inhibitor. It binds strongly to tissue kallikrein but weakly to other serine proteinases such as chymotrypsin and elastase. The tissue distribution and changes in kallistatin levels in human diseases were characterized by using specific monoclonal and polyclonal antibodies against kallistatin. Kallistatin antigen levels in blood cells, fluids, and tissues measured with a specific enzyme-linked immunosorbent assay showed displacement curves that were parallel with those in purified kallistatin, indicating their immunologic identity. Expression of kallistatin mRNA in platelets, neutrophils, lymphocytes, monocytes, endothelial cells, hepatocytes, and colon and prostate carcinoma cells was identified by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Plasma kallistatin concentration was 22.1 +/- 3.5 micrograms/ml in 30 normal subjects and 21.1 +/- 3.8 micrograms/ml in 5 patients with C1 inhibitor deficiency. A significantly reduced kallistatin level (7.2 +/- 2.5 micrograms/ml, p < 0.001) was seen in plasma samples from 9 patients with liver disease and 10 patients with sepsis (7.7 +/- 3.5 micrograms/ml, p < 0 .001). Further, kallistatin levels in 10 women taking oral contraceptives (19.8 +/- 3.8 micrograms/ml) and 21 pregnant women (14.9 +/- 3.3 microg/ml) were significantly lower than those seen in healthy individuals. These data suggest that kallistatin is found in plasma, is produced mostly in the liver, and can be consumed during sepsis. Its consumption in sepsis may indicate a protective role to prevent blood pressure lowering.