Role Of Cyclooxygenase-2 In The Development Of Portal Hypertension In Experimental Cirrhosis

Part OneEffect of rofecoxib, a selective COX-2 inhibitor, on the portal pressure in the CCl₄ induced cirrhotic ratsBackground & Aims: Cyclooxygenase (COX) is a key enzyme in the synthesis of prostanoids. Two isoforms (COX-1, COX-2) of this enzyme have been identified. Recent studies have suggested that COX-2 is up-regulation increase in human and experimental cirrhosis, suggesting a major role for COX-2 in the development of portal hypertension in cirrhosis. The aim of this study is to investigate whether COX-2 contributes to portal hypertension and to determine the effect of the selective COX-2 inhibitor, rofecoxib, on the portal pressure during the development of cirrhosis induced by CCl₄ in rats.Methods: Male S-D rats were used. Cirrhosis was induced by CCl₄(CCl₄/olive oil: v/v=1/1) twice a week for 8 weeks. Controls rats received olive oil (i.p.) only; CCl₄ induced rats were divided into three groups: one concurrently received placebo (saline solution by gavage), the others received rofecoxib (10mg · kg ^-1 · d^-1 by gavage) and misoprostol (10μg · d^-1 by gavage) for 8 weeks respectively. After 8 weeks portal pressure (PP) was measured, while the levels of PGE₂ and thromboxane B₂ (TXB₂) in the liver tissues were determined by enzyme immunoassay. The expression of COX-2 was assessed by Western blot and liver histopathological analysis was performed by using HE staining.Results: Rofecoxib treatment caused significant amelioration of portal hypertension relative to placebo treated rats (respective 11.95±1.05mmHg vs. 13.45 ± 1. 15 mmHg; R<0. 05), and also liver injury (reduced activities of serum aspartate aminotransferase and alanine aminotransferase). At the same time, rofecoxib treatment reduced PGE₂ and TXB₂ in liver tissue compared with placebo group (PGE₂:653.3 ± 198.7 vs. 870. 3 ± 230. 1, R<0. 05; TXB₂: 262.2 ± 77.7 vs. 352.8 ± 72. 1 , R<0. 05),although both of the COX-derived products still higher than normal control rats(TXB2:82. 5 + 51.2, PGE,:442. 4 + 165. 2). While portal pressure in PGE, group was unchanged compared to placebo (respectively 14.00+1. 12 mmHg vs. 13.45+1. 15 mmHg), and also in TXB2 and PGE2 in liver tissue(TXB2: 354.6 + 68. 5 vs. 352. 8 + 72. 1, P>0. 05; PGE2: 855. 3 ± 293. 9 vs. 870. 3 ± 230. 1, P>0. 05).Conclusions: The results emphasize the role of COX-2 in the development of portal hypertension and strongly suggest that blockade of its actions could be amelioration of portal hypertension. The decrease in portal pressure may at least account for the decreasing the COX-2 -derived products in liver tissue.Part TwoEffect of COX-2 on liver fibrogenesis and sinusoidal capillarizationinduced by CC14 in ratsBackground & Aims: The initial event in the pathophysiology of portal hypertension is increased vascular resistance to portal flow, primarily caused by structural changes such as the formation of fibrotic scar tissue, regenerative nodules compressing portal and central venues. Moreover, it has been shown that the capillarization of hepatic sinusoids also result in the increased vascular resistance. The hepatic sinusoidal capillarization results either from the development of neo-vessels or from the alteration of pre-existing sinusoids, both may be induced by the stimulation of pro-angiogenic factors. Recent evidence demonstrated that the production of prostanoids by COX-2 promotes the expression of pro-angiogenic factors. This raised the question of whether COX-2 and it's metabolites may involved in the formation of portal hypertension and the establishment of sinusoidal capillarization in cirrhotic liver. The aimof the investigation was, therefore, to investigate the effect of rofecoxib, a highly selective COX-2 inhibitor, and misoprostol on liver fibrogenesis and sinusoidal capillarization in cirrhotic rats.Methods: Induction of liver cirrhosis and study protocol described as above. Histopathological examination was performed using a histological scoring system, liver fibrogenesis was also assessed by digital image analysis of Masson's trichrome stained sections. Sinusoidal ultrastructure observed by transmission electron microscopy. The basement membrane proteins (collagen type IV, laminin ) levels and its localization in liver were determined by Western blot and immunohistochemistry, respectively. Numbers of activated hepatic stellate cells were quantified after alpha-smooth muscle actin (alpha-SMA) staining, and the microvessel density was detected following vWF (von Willebrand factor) immunolabel ing on liver tissue sections.Results: (1) Livers from CCl4-treated rats exhibited substantial damage, including fibrosis, inflammation, and extensive expression of smooth muscle alpha-actin (alpha-SMA). Compared to controls, rats administered rofecoxib revealed significant decrease in the degrees of fibrosis (fibrosis score respectively, 2.89 + 0.60 vs. 4. 00 + 0. 00, FKO. 05) and inflammation scores (1.0 + 0.5 vs. 1. 7 + 0. 5, K0. 05), and the fibrotic area was significantly higher in the control groups than rofecoxib group (30. 7 + 8. 9 vs. 23. 5±6. 5, K0. 05). Compared to placebo, administration of rofecoxib resulted in a marked decrease in the number of alpha-SMA-positive cells (respectively, 12.5 + 1.3 vs. 7.4+1.4, K0.01), while PGE, did not change fibrosis score or the number of alpha-SMA-positive cells (12.3+1.1 vs. 12. 5+1.3, P>0. 05). (2) In cirrhotic model rat, electron microscopy showed: accumulation of extracellular matrix proteins in the Disse's space; loss or reduction in number of sinusoidal endothelial fenestrate; development of subendothelial basal lamina (basement membrane formation). But those were ameliorated by administration of rofecoxib. (3) Fibrotic liver had significantly higher microvessel density compared with normal liver. Compared with placebo, rofecoxib resulted in significant decrease in microvessel density (respectively , 11.3 + 1.6 vs. 6.4 + 0.7, R0. 01) while PGE, did notchange it (respectively, 11.1 + 1.0 vs. 11.3 + 1.6, P>0.05). (4) Western blot analysis confirmed that laminin and type IV collagen significantly increased in CC14 induced cirrhotic rats compared with normal rats, but rofecoxib reduced both of laminin and type IV collagen levels compared with the model control rats which treated by CC14 plus saline.Conclusions: The early and chronic administration of rofecoxib may inhibit liver fibrogenesis and ameliorate sinusoidal capillarization in CC14 induced cirrhotic rats.Part ThreeEffect of rofexcoxib, a highly selective COX-2 inhibitor, and PGEi on the expression of VEGF and CTGF in cirrhotic liverBackground & Aims: Recently, it has been shown that vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) are important pro-angiogenic factors. This raised the question of whether VEGF and CTGF are downstream molecular of COX-2 pathway involved in the formation of hepatic fibrogenesis and the establishment of sinusoidal capillarization. So, the aims of this study is to further assess whether the administration of rofecoxib ameliorates hepatic fibrogenesis and sinusoidal capillarization by reduced VEGF and CTGF synthesis during the cirrhosis inductionMethods: Induction of liver cirrhosis and study protocol described as above. The connective tissue growth factor (CTGF) and Vascular endothelial growth factor (VEGF) proteins levels and its localization in livers were determined by Western blot and immunohistochemistry, respectively.Results: Compared to placebo, the proteins expressions of VEGF and CTGF were also significantly suppressed by rofecoxib treatment, while PGE, unchanged VEGF protein level in liver and decreased CTGF protein level...