Coupling Factor 6 Is Upregulated in Monocrotaline-induced Pulmonary Arterial Hypertension in Rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare and fatal disease. The prostacyclin (PGI2) pathway is a well-known therapeutic target for PAH. As a novel vasoconstrictive peptide, coupling factor 6 (CF6) has been recognized as the only endogenous inhibitor of PGI2. However, the relationship between CF6 and PAH is still unknown. In this study, we investigated the involvement of CF6 in PAH in rats. METHODS: A total of 80 Sprague-Dawley rats were randomly divided into 2 groups: a control group (with saline intraperitoneally) and a monocrotaline (MCT)-treated group (with MCT 60mg/kg intraperitoneal injection). The expression level of CF6 was measured by immunohistochemistry, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The plasma level of 6-keto-PGF1α, a stable metabolite of PGI2, was measured by enzyme-linked immunosorbent assay. RESULTS: After MCT injection, although the plasma level of CF6 was markedly increased in a time-dependent manner, the level of 6-keto-PGF1α was decreased in the MCT rats as compared to that in the controls (P < 0.05). Reverse transcription polymerase chain reaction indicated that the lung tissue level of CF6 messenger RNA (mRNA) in the MCT rats was significantly upregulated compared to the level in the controls. There was an intense CF6 immunostain concentrated on the lung tissue of the MCT rats compared with the controls. Univariate analysis indicated that the plasma level of CF6 was not correlated with blood pressure, fasting blood glucose, cholesterol (high-density lipoprotein, triglycerides and total cholesterol) or C-reactive protein level. CONCLUSIONS: These results demonstrated that CF6 was elevated in MCT-induced PAH rats and may play an important role in the development of PAH.

Novel pro-atherogenic molecule coupling factor 6 is elevated in patients with stroke: a possible linkage to homocysteine.

BACKGROUND AND PURPOSE: Homocysteine (Hcy) is an independent predictor of stroke. Coupling factor 6 (CF6) is regulated by nuclear factor kappa B (NF-kappaB) signaling which is activated by Hcy. We tested the hypothesis that CF6 is elevated with Hcy in stroke. We also examined the effect of vitamin treatment on CF6 and Hcy levels. METHODS AND RESULTS: The 59 Japanese patients with a recent history of stroke were randomly assigned to a group without vitamin treatment (Group 1, n = 29) and to a group with treatment with both folic acid and vitamin B(12) for 2 months (Group 2, n = 30). The CF6 level was elevated in the patients with stroke compared with that in controls (n = 64) at admission. In a multiple regression model, the plasma CF6 level was weakly correlated to the total Hcy (tHcy) level. In Group 1, the plasma tHcy and CF6 levels were unchanged. In Group 2, however, they were both decreased, and there was a weak positive correlation between the decreases in plasma levels of CF6 and tHcy. CONCLUSION: CF6 is elevated in patients with stroke independently of risk factors. Since Hcy and vitamin treatment affect CF6 levels in stroke, CF6 appears to be a novel molecule for the pathogenesis and treatment of stroke.

Plasma level of mitochondrial coupling factor 6 increases in patients with coronary heart disease.

BACKGROUND: The aim of the present study was to investigate alterations in the plasma level of coupling factor 6 (CF6), a novel endogenous inhibitor of prostacyclin, in patients with coronary heart disease. METHODS AND RESULTS: In total, 35 patients with coronary heart disease and 20 age-matched healthy subjects were examined. Plasma levels of CF6 and 6-keto-prostaglandin (PG)F(1a) (a stable metabolite of prostacyclin) were measured using radioimmunoassay. The plasma level of CF6 was significantly increased in patients (254.1+/-29.8 pg/ml vs 219.4 +/-36.7 pg/ml in controls, p<0.0001), whereas that of 6-keto-PGF(1a) was significantly decreased (23.4 +/-2.3 pg/ml vs 26.1+/-4.5 pg/ml in controls, p=0.001). Moreover, after percutaneous transluminal coronary angioplasty (PTCA) and stent therapy, the level of CF6 was further increased by 30% to 330.4+/-26.0 pg/ml, and that of 6-keto-PGF (1a) was decreased by 42% to 13.5+/-2.0 pg/ml, compared with baseline (all p<0.01). Univariate analysis showed a significant result that the plasma level of CF6 was inversely correlated with that of 6-keto-PGF(1a) in the patients. The plasma ratio of CF6 to 6-keto-PGF(1a) was 8.4 in the control group and that in patients with coronary heart disease was increased to 24.4 after the therapy from 10.9 before therapy. CONCLUSIONS: The results suggest that an increased CF6 level may be responsible in part for the decreased prostacyclin level observed in patients with coronary heart disease, in particular after PTCA and stent therapy. As a potential risk factor for coronary heart disease, CF6 might have important clinical significance.

Plasma level of mitochondrial coupling factor 6 increases in patients with type 2 diabetes mellitus.

Coupling factor 6 (CF6) is a novel endogenous inhibitor of prostacyclin. Plasma CF6 and 6-keto-PGF(1a) were measured by radioimmunoassay in 70 consecutively recruited patients with type 2 diabetes and in 56 healthy controls. A significantly increased plasma CF6 level was found in diabetics compared with controls. The CF6 level was inversely correlated with plasma 6-keto-PGF(1a) level and positively correlated with blood glucose and lipids. These results suggest that CF6 might be an obvious marker of impaired endothelium and might contribute to vascular damage in diabetes.

Tumor necrosis factor alpha as an endogenous stimulator for circulating coupling factor 6.

OBJECTIVE: We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the generation and release of CF6. METHODS AND RESULTS: We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-alpha enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-alpha in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I kappa B kinase alpha whose efficacy was confirmed by blockade of translocation of NF-kappa B p65 protein and of degradation of I kappa B alpha protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-alpha in a dose-dependent manner. CONCLUSIONS: TNF-alpha stimulates the gene expression of CF6 via activation of NF-kappa B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.

Plasma mitochondrial coupling factor 6 in patients with acute myocardial infarction.

Previous studies have shown that mitochondrial coupling factor 6 (CF6) is an endogenous peptide that inhibits prostacyclin (PGI2) synthesis in vascular endothelial cells. In this study, we measured the plasma CF6 level of patients with acute myocardial infarction (AMI) to observe dynamic changes of CF6. All patients showed elevated plasma CF6 levels upon admission for treatment of AMI. Their CF6 levels peaked approximately 72 h after the onset of AMI and remained high for 7 days. At 7 days, their CF6 levels decreased to the level seen upon admission, but not to within a normal range. Hyperlipidemic patients had significantly greater CF6 levels at 24 h after onset of AMI than patients with a normal lipid profile. On admission, the plasma CF6 level in patients with a cardiac function of Killip class > or =II was higher than that in patients with a Killip class I cardiac function. At 3 days after the onset of AMI, the plasma CF6 levels of patients with a creatinine kinase (CK) peak value > or =1,500 units/l were significantly higher than those of patients with a CK peak value <1,500 units/l (p =0.05). At 7 days after the onset of AMI, the plasma CF6 levels of patients who received no reperfusion were significantly higher than those of patients who received a successful reperfusion. The plasma CF6 levels of AMI patients at admission, at 24 h, and at 3 days after onset of symptoms correlated positively with the cardiac function by Killip classification, respectively. At 24 h after onset of AMI, the plasma CF6 levels correlated positively with plasma total cholesterol levels and low-density lipoprotein levels. At 3 days, the plasma level of CF6 correlated positively with the plasma CK peak value and correlated negatively with left ventricular ejection fraction. These results suggest that the plasma CF6 level was elevated in patients with AMI.

Circulating coupling factor 6 in human hypertension: role of reactive oxygen species.

OBJECTIVE: Coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and might function as an endogenous vasoconstrictor in the fashion of a circulating hormone in rats. We investigated the role of coupling factor 6 in human hypertension. METHODS AND RESULTS: The patients with essential hypertension (EH) (n = 30) received a series of normal salt diet (12 g salt/day) for 3 days, low salt diet (2 g salt/day) for 7 days, and high salt diet (20-23 g salt/day) for 7 days. Normotensive control subjects (n = 27) received normal and low salt diets. The plasma level of coupling factor 6, measured by radioimmunoassay, during normal salt diet was higher in patients with EH than in normotensive subjects (17.6 +/- 1.7 versus 12.8 +/- 0.5 ng/ml, P < 0.01). Whereas the plasma level of coupling factor 6 was unchanged after salt restriction in normotensive subjects, it was decreased after salt restriction (from 12 g/day to 2 g/day) and was increased after salt loading (from 2 g/day to 20-23 g/day) in patients with EH. This increase in plasma level of coupling factor 6 was abolished by oral administration of ascorbic acid, but the level of blood pressure was unaffected. The percentage changes in plasma coupling factor 6 level after salt restriction and loading were positively correlated with those in mean blood pressure (r = 0.57, P < 0.01), and negatively correlated with those in plasma nitric oxide level (r = -0.51, P < 0.05). CONCLUSION: These indicate that circulating coupling factor 6 is elevated in human hypertension and modulated by salt intake presumably via reactive oxygen species.

Plasma concentration of coupling factor 6 and cardiovascular events in patients with end-stage renal disease.

BACKGROUND: Plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of overall mortality and cardiovascular outcome in hemodialysis patients. However, not only ADMA but also traditional risk factors account for only part of the high cardiovascular morbidity and mortality in these patients. We investigated cross-sectionally the association between coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, and cardiovascular events in 95 hemodialysis patients. METHODS: Plasma CF6 level was measured by radioimmunoassay, whereas plasma ADMA level by high-performance liquid chromatography (HPLC). RESULTS: Plasma levels of CF6 and ADMA were threefold higher in hemodialysis patients than in control individuals, and there was a positive correlation between these two compounds (r=0.25, P < 0.05). Plasma CF6 level was positively correlated with serum creatinine level (r=0.36, P < 0.01) and was reduced after dialysis (P < 0.05). Plasma CF6 and ADMA levels were both higher in hemodialysis patients complicating ischemic heart disease (myocardial infarction and/or angina) than in those free of cardiovascular events. In a multiple regression model, plasma CF6 level (r=0.24, P=0.023) and ADMA level (r=0.26, P=0.023) were independently related to the occurrence of ischemic heart disease in hemodialysis patients. CONCLUSION: CF6 is a novel risk factor for ischemic heart disease in end-stage renal disease (ESRD). Synergism of this peptide and ADMA might contribute to its occurrence presumably by inhibition of prostacyclin and nitric oxide production. A prospective study is needed to evaluate this issue more precisely.

Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor.

We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.

The pharmacokinetics of trilostane and ketotrilostane in an interconverting system in the rat.

The pharmacokinetics of trilostane and one of its metabolites ketotrilostane are described and characterized in the rat following the separate intravenous administration of trilostane and ketotrilostane. It was noted during these studies that the parent compound and its metabolite undergo metabolic interconversion-trilostane producing ketotrilostane and ketotrilostane generating trilostane. This result means that trilostane is conserved in the body by interconversion--being metabolized to ketotrilostane and then subsequently back to the "parent" drug, trilostane.