Tumor necrosis factor ? regulates nitric oxide synthase expression in portal hypertensive gastric mucosa of rats

Abstract

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. 1-3 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. [4][5][6][7][8][9] 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

Activation of eNOS in rat portal hypertensive gastric mucosa is mediated by TNF-α via the PI 3-kinase-Akt signaling pathway

“…12 Second, the increased TNF-␣ likely increases the permeability of the PHT microvasculature and may also be involved in the morphologic changes resulting in the microvascular abnormalities of PHT gastropathy. 13 Furthermore, the role of TNF-␣ and NO overproduction in the increased susceptibility to injury is indicated by the fact that anti-TNF-␣ antibody and Nnitro-L-arginine methyl ester (L-NAME), respectively, reversed the increased susceptibility to injury produced by ethanol. 12,13 Although gastric TNF-␣ is elevated in portal vein-stenosed rats, the mechanism of this change remains unclear because it is well established that plasma TNF-␣ concentrations are normal in portal hypertension and increase in relation to the severity of liver disease.…”

“…13 Furthermore, the role of TNF-␣ and NO overproduction in the increased susceptibility to injury is indicated by the fact that anti-TNF-␣ antibody and Nnitro-L-arginine methyl ester (L-NAME), respectively, reversed the increased susceptibility to injury produced by ethanol. 12,13 Although gastric TNF-␣ is elevated in portal vein-stenosed rats, the mechanism of this change remains unclear because it is well established that plasma TNF-␣ concentrations are normal in portal hypertension and increase in relation to the severity of liver disease. However, the fact that TNF-␣ levels are elevated in pa- 44 This requirement also renders eNOS calcium dependent.…”

“…Eight SO and 8 PHT rats received anti-TNF-␣ neutralizing antibody (Genzyme, Cambridge, MA), and the remaining 8 SO and 8 PHT rats received vehicle intravenously at days 11 and 13 after the surgery according to a protocol previously reported. 13 The antibody (125 L) was diluted 1:3 with 10 mmol/L phosphate-buffered saline and was administered intravenously to each animal once daily. It has been shown that 1 mL of this antibody blocks at least 1,280 units of recombinant TNF-␣ and that after injection of 125 L of the antibody to the PHT rats, TNF-␣ is completely inhibited for 48 hours.…”

“…It has been shown that 1 mL of this antibody blocks at least 1,280 units of recombinant TNF-␣ and that after injection of 125 L of the antibody to the PHT rats, TNF-␣ is completely inhibited for 48 hours. 13 Twenty-four hours after the last injection, rats were anesthetized. After laparotomy, gastric tissues were excised and frozen in liquid N 2 for subsequent experiments.…”

“…Our previous study showed that elevated TNF-␣ up-regulates eNOS expression and its enzymatic activity in PHT gastric mucosa and that neutralization of TNF-␣ reverses the hemodynamic abnormalities of PHT gastric mucosa. 13 Although these data clearly indicate that TNF-␣ is involved in the activation of eNOS in PHT gastric mucosa, the molecular mechanisms and signaling pathways of eNOS activation and the role(s) of TNF-␣ in this signaling remain unknown.In general, NO generated by eNOS plays a pivotal role in maintaining vascular tone and angiogenesis. 16 …”