Celecoxib and octreotide synergistically ameliorate portal hypertension via inhibition of angiogenesis in cirrhotic rats
Abnormal angiogenesis is critical for portal hypertension in cirrhosis. Except for etiological treatment, no efficient medication or regime has been explored to treat the early stage of cirrhosis when angiogenesis is initiated or overwhelming. In this study, we explored an anti-angiogenesis effort through non-cytotoxic drugs octreotide and celecoxib to treat early stage of cirrhotic portal hypertension in an animal model. Peritoneal injection of thioacetamide (TAA) was employed to induce liver cirrhosis in rats. A combination treatment of celecoxib and octreotide was found to relieve liver fibrosis, portal venous pressure, micro-hepatic arterioportal fistulas, intrahepatic and splanchnic angiogenesis. Celecoxib and octreotide exerted their anti-angiogenesis effect via an axis of cyclooxygenase-2/prostaglandin E2/EP-2/somatostatin receptor-2, which consequently down-regulated phosphorylation of extracellular signal-regulated kinase (p-ERK)–hypoxia-inducible factor-1α (HIF-1α)–vascular endothelial growth factor (VEGF) integrated signaling pathways. In conclusions, combination of celecoxib and octreotide synergistically ameliorated liver fibrosis and portal hypertension of the cirrhotic rats induced by TAA via the inhibition of intrahepatic and extrahepatic angiogenesis. The potential mechanisms behind the regimen may due to the inactivation of p-ERK–HIF-1α–VEGF signaling pathway.
KeywordsAngiogenesis Celecoxib Octreotide Portal hypertension Hepatic arterioportal fistulas Liver cirrhosis
Vascular endothelial growth factor
Platelet-derived growth factor BB chain
Alpha smooth muscle actin
Masson’s trichrome staining
Hematoxylin and eosin staining
Micro-hepatic arterioportal fistulas
Extracellular signal-regulated kinase
Human umbilical vein endothelial cells
Dulbecco’s modified Eagle’s medium
Quantitative real-time PCR
Nonsteroidal anti-inflammatory drugs
Portal hypertension represents the leading cause of mortality and liver transplantation in patients with cirrhosis. Increased intrahepatic vascular resistance and portal venous blood flow are supposed as the major pathological progresses in development of portal hypertension . As a clinical treatment, transjugular intrahepatic portosystemic shunt is the best way to decrease the structural intrahepatic resistance. To reduce the splanchnic blood flow, nonselective β-blocker and vasoactive drugs are widely used. However, all of those modalities are the last resort for the advanced or decompensated cirrhotic patients . Likewise, new medication or regime should be explored to treat the early stage of cirrhosis when angiogenesis is initiated [3, 4, 5].
Cyclooxygenase-2 (COX-2), a rate-limiting enzyme involved in the conversion of arachidonic acid to prostaglandins and thromboxanes, is up-regulated in cirrhotic liver . It has been demonstrated that COX-2 inhibitors could inhibit angiogenesis in hepatocellular carcinoma (HCC), breast cancer and other solid tumors [7, 8, 9, 10, 11]. Moreover, our recent studies revealed that inhibition of COX-2 through a selective inhibitor celecoxib ameliorates portal hypertension in an animal model [12, 13]. Moreover, such effect is related to its inhibition of intrahepatic angiogenesis and epithelial-to-mesenchymal transition of hepatocytes [12, 13]. Somatostatin (SST) and its analogue octreotide are widely used for the management of bleeding from gastroesophageal varices in patients with cirrhosis . It is notable that octreotide could inhibit angiogenesis in HCC and in the early stage of portal hypertension induced by partial portal vein ligation [15, 16]. Our previous studies have shown that the combination of COX-2 inhibitor and somatostatin analogue synergistically enhanced the anti-angiogenesis effect in HCC [16, 17]. In this study, we aimed to investigate the effects of celecoxib in combination with octreotide on portal hypertension, and intrahepatic and extrahepatic angiogenesis. The potential mechanisms behind the regimen were also investigated through in vivo and in vitro experiments.
Materials and methods
Animals and grouping
Sprague–Dawley rats weighing 200–250 g were obtained from Experimental Animal Center of Sichuan University (Chengdu, China). The animal procedures were approved by the Animal Use and Care Committee of Sichuan University and were conducted according to regulations set by Sichuan University. Peritoneal injection (i.p.) of thioacetamide (TAA, Sigma-Aldrich, St. Louis, MO, USA) was used to induce liver cirrhosis (200 mg/kg every 3 days for 16 weeks). 60 rats were randomized into control, TAA and TAA+ combination groups with 20 rats in each group. Control group received normal saline (1 mL i.p., every 3 days); TAA group received TAA; TAA+ combination group received TAA plus celecoxib (gastric gavage, 20 mg/kg/day, Pfizer, New York, NY, USA) and octreotide (intramuscular injection, 50 μg/kg/day, Novartis, Basel, Switzerland). Celecoxib and octreotide were given from the initiation of TAA administration.
Methods for hemodynamic measurements, histopathological evaluation, vascular casting, quantitative real-time PCR (qRT-PCR), immunohistochemistry staining, Western blot, enzyme-linked immunosorbent assay (ELISA), ink-gelatin-dextran perfusion for hepatic arterioportal fistulas (hAPF), Cell culture and treatments, immunocytofluorescence staining, wound-healing assay, tube formation assay and chromatin immunoprecipitation assay (ChIP) are described in the supporting information.
All data were expressed as mean ± SD and were analyzed by SPSS 19.0 software (SPSS, Chicago, IL, USA). For multi-group comparison, one-way ANOVA followed by SNK multiple comparison test was implemented. Natural Log transformation was utilized to transfer non-normal distribution variables into normal distribution variables. A value of p < 0.05 was considered significant.
Amelioration of liver fibrosis by the combination treatment
Reduction of portal hypertension by the combination treatment
There were no significant differences of the mean arterial pressure and heart rate among three groups (p > 0.05, Fig. 1f, g). A significant increase of portal venous pressure in the TAA group by 60.9 % was noticed compared with that in the control group. However, the portal venous pressure decreased substantially in the TAA+ combination group (p < 0.05, Fig. 1h).
Inhibition of hepatic angiogenesis by the combination treatment
Reduction of micro-hepatic arterioportal fistulas (micro-hAPF) by the combination treatment
Ink perfusion assay detected micro-hAPF in TAA and TAA+ combination groups, but not in the control group (Supporting Fig. S1A). Compared with that in the TAA group, the average number of micro-hAPF per liver in the TAA+ combination group reduced significantly (p < 0.05, Supporting Fig. S1B).
Suppression of splanchnic angiogenesis by the combination treatment
Inhibition of the signal pathways related to intrahepatic angiogenesis by the combination treatment
Suppression of the axis of COX-2/PGE2/EP-2/p-ERK/VEGF by celecoxib
The protein and mRNA levels of intrahepatic COX-2, as well as serum concentration of prostaglandin E2 (PGE2) in the TAA group, were found to significantly increase compared with those in the control group. However, the suppression of COX-2 and PGE2 was revealed in the TAA+ combination group (p < 0.05, Supporting Fig. S5A-D).
Inhibition of the cascade of SST/SSTR-2/p-ERK/VEGF by octreotide
The mRNA levels of SSTR-1, SSTR-2 and SSTR-5 but not SSTR-3 or SSTR-4 were significantly up-regulated in the livers of the TAA group when compared with those in the control group (Supporting Fig. S6A). SSTR-2 and SSTR-5 could be detected in HUVEC (Supporting Fig. S6B).
Synergistical inhibition of p-ERK–HIF-1α–VEGF by celecoxib and octreotide in vitro
Prevention of HIF-1α binding to VEGF promoter with the combination treatment
In IgG negative control, no HIF-1α binding to VEGF promoter in all of treatments could be measured (Fig. 7i). Conversely, using anti-HIF-1α antibody, we detected recruitment of HIF-1α in the VEGF promoter by the cells treated by DMSO (9.06 ± 1.07 % of the input), indicating activation of the pathway and activation of VEGF transcription. However, the recruitments of the transcription factor were significantly reduced after the treatments in the following levels: celecoxib (3.21 ± 0.63 %), octreotide (6.06 ± 0.97 %), celecoxib + octreotide (1.41 ± 0.26 %) and AZD6244 (2.08 ± 0.32) (p < 0.05, Fig. 7i).
In the cirrhotic liver, proliferated cells (hepatic stellate cells and fibroblasts) require sufficient blood flow and nutrition to sustain their proliferation status. Consequently, angiogenesis is enhanced in response to a variety of proangiogenic stimuli . Accumulated data reveal that intrahepatic angiogenesis is driving force of portal hypertension due to a tortuous vascular network of varying diameter and flow pattern which was organized into micronodules and macronodules, enhancing intrahepatic vascular resistance [1, 4]. Moreover, increased vascular bed size of the portal venous system mediated by angiogenesis significantly contributes to increased portal venous blood flow . Our study observed not only intrahepatic but also extrahepatic angiogenesis in the process of portal hypertension in a cirrhotic rat model induced by TAA. Moreover, it was speculated that extensive intrahepatic angiogenesis might lead to the formation of micro-hAPF, an intrahepatic communication between the hepatic artery and the portal venous system. Micro-hAPF would exacerbate portal hypertension by introducing a tremendous arterial blood flow into the intrahepatic portal venous system. To our knowledge, it was the first time to visualize the supposed formation of micro-hAPF in this study. Such confirmation suggests the crucial role of effective anti-angiogenesis in prevention or treatment of liver cirrhosis. It is gratifying that celecoxib in combination with octreotide not only arrested intrahepatic and extrahepatic angiogenesis but also reduced the formation of micro-hAPF synergistically. As a result, liver fibrosis was impressively ameliorated and the portal venous pressure decreased by 34 %.
VEGF is a pivotal regulatory protein in angiogenesis. The up-regulation of VEGF, VEGF receptor-2 and PDGF-BB has been verified in animal models of portal hypertension and liver fibrosis [19, 20, 21]. The antagonists of VEGF receptor 2, such as Sorafenib, Sunitinib, and combination of Imatinib and Rapamycin may decrease portal hypertension and ameliorate liver fibrosis through the anti-angiogenesis [20, 22, 23]. Similar to Sorafenib, Sunitinib and Imatinib, which inhibit angiogenesis via blockade of VEGF signaling pathway by decreasing VEGF receptor-2, or VEGF [20, 22, 23, 24], inhibition of angiogenesis in liver and intestine was also observed when celecoxib was combined with octreotide to suppress VEGF in this study. Anti-angiogenesis strategy was wildly used in the metronomic chemotherapy of solid cancer. A few angiogenesis inhibitors have been tested or approved in the treatment of HCC , but they are not recommended for patients with liver cirrhosis partly because of their toxicities and side effects [26, 27].
Consistent with previous reports [19, 20, 21, 28], the angiogenesis was associated with up-regulated proangiogenic molecule (VEGF, PDGF-BB and FGF-2) in cirrhotic liver induced by TAA in this study. Moreover, it has been reported that HIF-1α/VEGF, endothelial nitric oxide synthase, MAPK-ERK, JNK-p38 and TGF-β1/Smads signaling pathway were involved in effect of celecoxib and octreotide on the anti-angiogenesis [15, 29]. The p-ERK–HIF-1α pathway was up-regulated in the cirrhotic liver of this study. Celecoxib in combination with octreotide led to down-regulation of p-ERK–HIF-1α pathway in vivo. This synergetic inhibitory effect of celecoxib and octreotide on p-ERK–c-Fos–HIF-1α was further verified in vitro. Among the integrated signal pathways regulating VEGF, the MAPK-ERK signaling pathway involves in the crosstalk of COX-2 and SST signal transduction [30, 31]. Blockade of MAPK-ERK signaling pathway, either by a MEK inhibitor AZD6244 or by combination of celecoxib and octreotide, not only led to significant inhibition of p-ERK, HIF-1α, VEGF and angiogenesis in vitro, but also prevented HIF-1α from binding to VEGF promoter.
PGE2, one of catalysates of COX-2, is involved in modulation of angiogenesis and fibrosis via its receptors . The over-expressions of COX-2 and PGE2 in cirrhotic liver were substantially suppressed by celecoxib plus octreotide in vivo. By using PGE2 and EP-2 inhibitor (AH6809), the experiment showed that celecoxib exerted its anti-angiogenesis effect via COX-2–PGE2–EP-2–p-ERK–VEGF pathway. The anti-angiogenetic effects of octreotide are usually mediated by SSTR-2, SSTR-3 and SSTR-5 [15, 30]. However, this study indicated that SSTR-2 was the only factor which octreotide predominantly activated.
Celecoxib has been widely used in the clinical treatment of osteoarthritis and rheumatoid arthritis . Several studies have demonstrated that celecoxib could efficiently ameliorate portal hypertension and fibrosis in several animal models [6, 12, 13, 34]. Octreotide is indicated for the management of bleeding from gastroesophageal varices in patients with cirrhosis . The regime to combine celecoxib with octreotide was firstly investigated in the rat model with cirrhotic portal hypertension in our study. Both drugs were administrated since the beginning of TAA treatment. Therefore, this regime might be beneficial to early stage of cirrhotic portal hypertension.
The main adverse reactions of nonsteroidal anti-inflammatory drugs (NSAIDs) are gastrointestinal and cardiovascular toxicities. However, celecoxib showed very low toxicity in a metronomic chemotherapy of mammary adenocarcinomas and advanced refractory gastrointestinal cancers [8, 9, 10, 35]. Moreover, compared with nonselective NSAIDs, celecoxib was associated with a low risk of clinically significant upper and/or lower gastrointestinal adverse events in patients with osteoarthritis or liver cirrhosis [36, 37].
This study is supported by Natural Science Fund of China (81170413 and 81400637), the Chinese Postdoctoral Science Foundation (2014M560721 and 2015T80984), Chinesisch-Deutsches Zentrum für Wissenschaftsförderung (GZ1065) and the Science and Technology Support Program of Sichuan province (2016SZ0041). The authors would like to thank Xian Li, Ou Qiang and Shu-Ping Zheng for their technical assistance. The authors also thank Prof. Yuan-Ping Han (College of Life Science, Sichuan University, Chengdu, China) and Dr. Wen-Han Yu (National Eye Institutes, National Institutes of Health, Baltimore, MD, USA) for the language modification of this article.
Jin-Hang Gao, Shi-Lei Wen, Wen-Juan Yang, Yao-Yao Lu and Shi-Hang Tang were involved in cellular and molecular biology experiments. Yao-Yao Lu, Zhi-Yin Huang and Shi-Hang Tang were in charge of experiment on animals. Shi Feng, Huang Tong, Ying-Mei Tang and Hui-Qi Xie performed immunohistochemical staining and histopathological evaluation. Shi Feng, Huang Tong and Jin-Hui Yang were responsible for statistical analysis. Jin-Hang Gao, Shi-Lei Wen, Rui Liu and Hui-Qi Xie were involved in ink perfusion, vascular casting and hemodynamic measurements. Zhi-Yin Huang carried out scanning electron microscope analysis. Jin-Hang Gao, Shi-Lei Wen and Cheng-Wei Tang wrote and revised the manuscript. Cheng-Wei Tang designed the concept and directed the research. Jin-Hang Gao and Cheng-Wei Tang obtained funding.
Compliance with ethical standards
Conflict of interest
All authors declare no conflict of interests.
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