Anti-tumor necrosis factor ␣ (TNF-␣) treatment decreases nitric oxide (NO) synthesis and ameliorates the hyperdynamic circulation in portal hypertensive rats. We have recently demonstrated that nitric oxide synthase isoform 3 (NOS3) is overexpressed in portal hypertensive gastric mucosa and that resultant NO overproduction probably is responsible for the increased susceptibility of the mucosa to damage. In the present study, we examined whether TNF-␣ is overexpressed in portal hypertensive gastric mucosa and whether anti-TNF-␣ treatment affects gastric NOS3 messenger RNA (mRNA) and protein expression. We examined plasma concentrations of TNF-␣ and its protein expression in gastric specimens from portal hypertensive and sham-operated rats using Western blotting and immunohistochemistry. We also measured gastric mucosal blood flow, gastric expression of NOS3 mRNA and protein, and NOS3 enzyme activity in rats with and without TNF-␣neutralizing antibody treatment. The TNF-␣ protein levels in portal hypertensive stomachs were significantly increased by 57% compared with levels in sham-operated controls. TNF-␣ antibody treatment normalized gastric mucosal blood flow in portal hypertensive stomachs and significantly reversed overexpression of gastric NOS3 mRNA, protein, and its enzyme activity in portal hypertensive rats by 48%, 45%, and 33%, respectively. These results suggest that TNF-␣ may regulate NOS3 expression in the portal hypertensive stomach and that anti-TNF-␣ treatment may ameliorate the pathophysiological abnormalities of portal hypertensive gastric mucosa. (HEPATOLOGY 1998;27: 906-913.)
Portal hypertensive (PHT) gastropathy is now recognized as a clinical entity that is frequently seen in patients with portal hypertension. The PHT gastric mucosa has distinct morphological and functional abnormalities that increase its susceptibility to damage by noxious factors such as alcohol, aspirin, and ischemia/reperfusion. We have recently showed that nitric oxide synthase isoform 3 (NOS3) messenger RNA (mRNA) and protein are overexpressed in PHT gastric mucosa of rats, and this overexpression may be, at least in part, responsible for the increased susceptibility of PHT gastric mucosa to damage. 10 However, the mechanisms that activate NOS3 in PHT gastric mucosa are unknown.
Tumor necrosis factor ␣ (TNF-␣) is a multifunctional cytokine that plays an important role in the pathogenesis of tissue damage seen in multiple unrelated conditions such as malaria, chronic heart failure, septic shock, and infectious diseases. 11 This cytokine is activated by various factors such as lipopolysaccharide, tissue injury, tumor cells, and hypoxia. 12,13 TNF-␣ increases vascular permeability and causes structural and metabolic changes in vascular endothelial cells in some tissues (e.g., lungs). It also enhances neutrophil attachment to umbilical vein endothelial cells, promoting the inflammatory response to tissue injury. However, its role in gastric mucosa under normal or pathological conditions remains unknown.
In patients with chronic liver diseases, the blood concentration of TNF-␣ and its receptor is elevated compared with concentrations in normal controls ; this elevation correlates with the severity of liver disease. In PHT rats, plasma concentrations of TNF-␣ are elevated. Lopez-Talavera et al. showed that anti-TNF-␣ neutralizing antibody significantly reduces the systemic hyperdynamic circulation and elevated portal venous pressure of PHT rats. Moreover, they used thalidomide, a selective inhibitor of TNF-␣, and showed that this treatment also decreases nitric oxide (NO) synthesis and ameliorates the hyperdynamic circulation of PHT rats. Thus TNF-␣ may be a major contributor to the hyperdynamic circulation of portal hypertension through activation of the NO/L-arginine pathway.
Expression and localization of TNF-␣ protein has not been defined in PHT gastric mucosa. In addition, it is not known whether TNF-␣ regulates NOS3 gene expression and enzyme activity in the PHT gastric mucosa or whether it modulates gastric mucosal blood flow in portal hypertension.
Because NOS3 enzyme activity is increased in the PHT vasculature and stomach, we postulated that overproduced TNF-␣ could regulate expression of NOS3 in PHT gastric mucosa. Therefore, our present study was aimed at Abbreviations: PHT, portal hypertensive; NOS3, nitric oxide synthase isoform 3 (endothelial constitutive isoform); TNF-␣, tumor necrosis factor ␣; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription; PCR, polymerase chain reaction; L-NAME, N -nitro-L-arginine methyl ester; NOS2, nitric oxide synthase isoform 2 (inducible isoform).