Potential conflict of interest: Nothing to report.
Liver cirrhosis is a very complex disease in which several pathological processes such as inflammation, fibrosis, and pathological angiogenesis are closely integrated. We hypothesized that treatment with pharmacological agents with multiple mechanisms of action will produce superior results to those achieved by only targeting individual mechanisms. This study thus evaluates the therapeutic use of the multitargeted receptor tyrosine kinase inhibitor Sunitinib (SU11248). The in vitro effects of SU11248 were evaluated in the human hepatic stellate cell line LX-2 by measuring cell viability. The in vivo effects of SU11248 treatment were monitored in the livers of cirrhotic rats by measuring angiogenesis, inflammatory infiltrate, fibrosis, α-smooth muscle actin (α-SMA) accumulation, differential gene expression by microarrays, and portal pressure. Cirrhosis progression was associated with a significant enhancement of vascular density and expression of vascular endothelial growth factor-A, angiopoietin-1, angiopoietin-2, and placental growth factor in cirrhotic livers. The newly formed hepatic vasculature expressed vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1. Interestingly, the expression of these adhesion molecules was adjacent to areas of local inflammatory infiltration. SU11248 treatment resulted in a significant decrease in hepatic vascular density, inflammatory infiltrate, α-SMA abundance, LX-2 viability, collagen expression, and portal pressure. Conclusion: These results suggest that multitargeted therapies against angiogenesis, inflammation, and fibrosis merit consideration in the treatment of cirrhosis. (HEPATOLOGY 2007.)
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According to reports of the World Health Organization, cirrhosis is one of the leading causes of mortality worldwide and a shortened life expectancy despite recent therapeutic advances. Cirrhosis is characterized by the presence of a hepatic inflammatory infiltrate that occurs early in the history of the disease, prior to the onset of significant clinical manifestations; it becomes chronic during the evolution of the illness, particularly in chronic viral hepatitis. 1 This dynamic inflammatory state is believed to contribute to the progression of liver fibrogenesis and cirrhosis. 2
Angiogenesis is of major importance during adult tissue repair 3 and is also a hallmark of inflammatory processes where both phenomena are closely integrated. For instance, many inflammatory mediators have direct angiogenic activities, and may also indirectly stimulate other cells to produce angiogenic factors such as vascular endothelial growth factor (VEGF)-A. 4 Angiogenesis, in turn, contributes to the perpetuation and the amplification of the inflammatory state due to the expression of adhesion molecules and chemokines in the neovasculature, which promote the recruitment of inflammatory cells. The inflammatory response can also be accentuated by new vessels acting as transporters of nutrients to the site of inflammation and tissue remodeling. 5 In this context, there is considerable evidence suggesting that during early inflammatory states, angiogenesis may contribute to the transition from acute to chronic inflammation.
The existence of important angiogenic processes in cirrhotic liver is widely accepted. For instance, in cirrhotic livers, scar tissue is surrounded by a dense vasculature. 6 Additionally, neovascularization is significantly increased during the development of liver fibrosis in both human and animal studies. 7 In light of these observations, and considering that chronic inflammation and angiogenesis are associated in many disorders, this link could also be exploited therapeutically in cirrhosis to modulate chronic inflammation.
An interesting therapeutic candidate to be used as an inhibitor of angiogenesis in cirrhosis is Sunitinib (SU11248). This indolinone molecule was designed to have a broad selectivity for the split kinase family of receptor tyrosine kinases, including KIT and FLT3, 8 and has efficacy as a potent antitumor and antiangiogenic agent in clinical trials for treatment of cancer. 9, 10 Its antiangiogenic efficacy is attributable to inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), both of which are essential for angiogenesis development. 11 Apart from angiogenesis, the inhibition of PDGFR has an additional therapeutic capability, because PDGF is among the most potent mitogenic and chemotactic agents for hepatic stellate cells (HSCs). 12 These cells, which rapidly induce the receptor β subunit (PDGFR-β) when they are activated, 13 play a key role in the development and progression of hepatic fibrosis.
We hypothesized that SU11248 treatment may have multiple mechanisms of action against cirrhosis progression. Therefore, we tested its efficacy in culture and in an animal model of cirrhosis.
The study was performed in male adult Wistar rats according to the criteria of the Investigation and Ethics Committees of the Hospital Clínic. Cirrhosis was induced by inhalation of CCl4 as described previously. 14 For the in vivo study, 10 cirrhotic rats were treated daily with 40 mg/kg of SU11248 (Pfizer, Inc., NY) via oral administration for 1 week. In parallel, another group of 10 cirrhotic rats received a daily intragastric administration of citrate-buffered solution (pH 3.5) as a control.
Fluorescein Isothiocyanate–Dextran Angiography, Histology, Immunohistochemistry, and Immunofluorescence.
See online expanded Experimental Procedures.
Reverse-Transcription Polymerase Chain Reaction and GeneChip Hybridization.
Total RNA was extracted from the frozen liver with Trizol reagent (Life Technologies, Rockville, MD). One microgram of total RNA was reverse-transcribed using the First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Then, complementary DNA samples were amplified for 30-35 cycles (94°C for 30 seconds, 55-60°C for 30 seconds, 72°C for 1 minute). Specific primers used for complementary DNA amplification were: placental growth factor, 5′-GTCGCTGTAGTGGCTGCTGTGGTG-3′ (forward primer) and 5′-TGTGGGGTTTTGCTTTGCTTCCTC-3′ (reverse primer); and HPRT, 5′-GGGGCTATAAGTTCTTTGCTGAC-3′ (forward primer) and 5′-CATTTTGGGGCTGTACTGCTTGAC-3′ (reverse primer).
RNA from livers of 4 nontreated cirrhotic rats and 4 SU11248-treated cirrhotic rats was hybridized to 8 high-density oligonucleotide microarrays (Affymetrix, Santa Clara, CA) as described previously. 14 Microarray results have been deposited in the Gene Expression Omnibus MIAME-compliant database (http://www.ncbi.nlm.nih.gov/geo/; accession number: GSE6929).
Cell Culture and Western Blotting.
Human umbilical vein endothelial cells were isolated and cultured as described previously. 15 The LX-2 cell line, which is derived from normal primary human HSCs that have been immortalized by selection in low serum, were maintained as described previously. These cells expressed markers of activated HSCs and presented a similar phenotype to that of activated HSCs in vivo. 16
Serum-starved cells were incubated with or without 1 μM SU11248, 100 ng/mL human VEGF-A, 200 ng/mL human angiopoietin-1, and 20 ng/mL PDGF-BB (R&D Systems, Minneapolis, MN). Then, cell lysates were prepared in lysis buffer and Western blotting was performed using anti-Erk-1/2 mitogen-activated protein kinase (MAPK), anti–phospho-Erk-1/2 (Thr202/Tyr204) MAPK, anti-Akt, and anti–phospho-Akt (Ser473)–specific antibodies (Cell Signaling Technology, Beverly, MA) as described previously. 17
Cell Viability Assay.
For the quantification of cell viability, we performed the MTT assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reagent (Sigma Chemical, St. Louis, MO). (See also online expanded Experimental Procedures available at http://interscience. wiley.com/jpages/0270-9 139/suppmat/index.html.)
Animals were anesthetized with inactine (0.5 mL/kg) and prepared for measurement of hemodynamic parameters as described previously. 17 Mean arterial pressure and portal pressure were continuously recorded in a multichannel system (MX4P and MT4; Lectromed, Ltd., Jersey, Channel Islands, UK).
Statistical differences were analyzed via Student t test. A P value of less than 0.05 was considered significant. Gene expression analysis was performed using libraries from the Bioconductor project (http://www.bioconductor.org) rooted in the computing environment R (see also online expanded Experimental Procedures).
Vascular Proliferation During Cirrhosis Progression.
Vascular proliferation was investigated via immunolabeling using the anti-vWF antibody and by fluorescein angiography. In cirrhotic livers, vWF-labeled vessels were nearly 10 times more prevalent than in control rats (Fig. 1A, panels b and a, respectively; Fig. 1C). Similarly, fluorescein isothiocyanate (FITC)–dextran angiography revealed significant growth of a disorganized vascular network in cirrhotic livers that was associated with the absence of normal lobular drainage through the central vein (Fig. 1A, panels d and c, respectively). Vascular growth was much more pronounced as cirrhosis progressed (data not shown). Next, vascular changes were also analyzed in other splanchnic beds, including the mesenteric region (Fig. 1B, panels e and f) and the tissue adjacent to the hepatic hilum (Fig. 1B, panels g and h). vWF immunolabeling showed an increase in the number of mesenteric vessels in cirrhotic rats (Fig. 1B, panels e and f; Fig.1D). These results were corroborated by FITC–dextran angiographies (Fig. 1B, panels g and h).
Induction of Angiogenic Factors in Cirrhotic Livers.
Liver tissue was processed from control and cirrhotic rats at 8-12 weeks (early cirrhosis) and 17-20 weeks (advanced cirrhosis), respectively, after the beginning of CCl4 treatment. All cirrhotic animals included in the second group had well-developed cirrhosis and ascites. There was a marked increase in the abundance of VEGF-A, angiopoietin-1 (ang-1), angiopoietin-2 (ang-2), and placental growth factor in cirrhotic livers compared with controls. The expression of these growth factors was higher in cirrhotic rats with ascites than in those without ascites (Fig. 2A).
Because the expression of VEGF-A and ang-2 is activated by hypoxia, we next investigated whether the distribution of these proteins may be attributable to hypoxic conditions. Immunohistochemistry for pimonidazole protein adducts has been used as a tissue marker of hypoxia. 18 As shown in Figure 2A, pimonidazole staining was not detected in control livers. In contrast, the distribution of pimonidazole adducts displayed a similar pattern to that of VEGF-A and ang-2 expression in cirrhotic livers.
Cellular Infiltration and Correlation with the Expression of VCAM-1 and ICAM-1 in the Vasculature of Cirrhotic Livers.
As shown in Fig. 3, CD11b-positive and CD3-positive cells were barely detected in livers of control rats. In contrast, a significant increase in CD11b and CD3 immunoreactivity was detected in cirrhotic livers. This increase in inflammatory infiltrate density was mainly localized in portal tracts and fibrous septa close to the newly formed vasculature. Next, the expression of VCAM-1 and ICAM-1 cell adhesion molecules in these newly formed blood vessels was examined with VCAM-1/ICAM-1 and vWF immunofluorescent staining (Fig. 4). VCAM-1 was weakly expressed in the sinusoidal lining cells of control livers and did not colocalize with blood vessels. In contrast, VCAM-1 immunostaining in cirrhotic livers was stronger and colocalized well with the neovasculature (yellow color in the merge panels). Similar to VCAM-1, a weak constitutive ICAM-1 expression was present in the hepatic sinusoid of control animals. In contrast, portal tracts and sinusoids of cirrhotic livers displayed a strong ICAM-1 immunoreactivity that colocalized with vWF immunostaining (yellow color in the merge panels).
SU11248 Inhibits VEGF-A and Ang-1–Dependent Activation of Erk-1/2 In Vitroand Angiogenesis In Vivo.
The activity of SU11248 against VEGFR and Tie2 receptors was evaluated in vitro. Stimulation of human umbilical vein endothelial cells with VEGF-A and ang-1 induced time-dependent activation of Erk-1/2 MAPK. This effect was inhibited by preincubation of human umbilical vein endothelial cells with 1 μM SU11248 (Fig. 5A).
Next, the antiangiogenic activity of SU11248 was evaluated in vivo via immunostaining with an anti-vWF antibody and via FITC–dextran angiography. As shown in Fig. 5B, cirrhotic rats treated with SU11248 (panel b) exhibited a significant reduction (P < 0.001) in hepatic vascularization compared with cirrhotic rats treated with vehicle (panel a) (5.1 ± 0.2 vessels/field versus 16.4 ± 0.4 vessels/field, respectively). This decrease in hepatic vascular density was also observed on FITC–dextran angiography (panels c and d). Similarly, the number of secondary vessel branches quantified in the small intestine was significantly lower (P < 0.001) in SU11248-treated cirrhotic animals (panel f) than in cirrhotic rats treated with vehicle (panel e) (2.8 ± 0.2 vessels/field versus 20.2 ± 1.7 vessels/field, respectively). No tertiary vessel branches were detected in the bowel of SU11248-treated animals compared with the significant number of tertiary branches detected in vehicle-treated cirrhotic animals. Furthermore, SU11248 treatment was also associated with a significant decrease in vessel density in the tissue adjacent to the portal vein (data not shown). These results demonstrated that the treatment of cirrhotic rats with SU11248 effectively inhibits angiogenesis in all of the tissues examined.
SU11248 Treatment Decreases Inflammatory Infiltrate in Cirrhotic Livers.
In agreement with Fig. 3, portal tracts of cirrhotic livers displayed a strong immunoreactivity for anti-CD11b and anti-CD3 antibodies (Fig.6, panels a and c, respectively) that was adjacent to vWF-positive areas (Fig. 6, panels e and g). After inhibition of angiogenesis by SU11248, a significant reduction in macrophages and lymphocyte infiltrate was detected in cirrhotic livers (Fig. 6, panels b and d and histograms i and j, respectively) which correlated well with the diminution of the vasculature detected in portal areas (Fig. 6, panels f and h). Similar results were obtained by quantifying CD68-positive cells as a measure of macrophage cell infiltration in cirrhotic livers (Supplementary Fig. 1).
Effect of SU11248 Treatment on HSC Activation, Fibrosis, and Portal Pressure.
Immunostaining demonstrated that many α-SMA–positive cells accumulated along the fibrous septa (Fig. 7A, panel a). Treatment with SU11248 drastically reduced the number of positive cells in cirrhotic livers as shown via immunohistochemistry and computer-assisted quantification (Fig. 7A, panel b; Fig. 7B). Next, liver fibrosis was assessed using Masson's trichrome stain. As expected, strong collagen staining was seen around the portal tracks and fibrotic septa in livers of cirrhotic rats (Fig. 7A, panel c). Importantly, cirrhotic rats treated with SU11248 showed a significant 30% decrease in hepatic collagen accumulation (Fig. 7A, panel d; Fig. 7C).
Next, the transcriptional modifications induced by SU11248 in cirrhotic livers were investigated. Gene expression was analyzed on high-density oligonucleotide microarrays. In agreement with Fig. 7A-C, SU11248 treatment induced a significant down-regulation of genes mainly related to HSC activation and extracellular matrix formation such as α-SMA, Col4α1, Col1α2, Col5α2, and Col1α1 (Supplementary Table 1).
The hemodynamic effect of SU11248 was evaluated through the measurement of mean arterial pressure and portal pressure in cirrhotic rats. As expected, cirrhotic rats had hypotension, a characteristic feature of cirrhosis (107 mm Hg). However, treatment with SU11248 did not modify mean arterial pressure (data not shown). In contrast, the SU11248-treated group exhibited a significant 40% decrease in portal pressure compared with nontreated cirrhotic rats (Fig. 7D).
To test the inhibitory effect of SU11248 treatment on PDGFR activation, we treated serum-starved LX-2 cells with PDGF-BB (20 ng/mL) for 0 to 30 minutes in the presence or absence of 1 μM SU11248. PDGFR-β activity was assessed by measuring activation-specific phosphorylation of Erk-1/2 MAPK and Akt via immunoblotting. As seen in Fig. 7E, PDGF-BB treatment markedly increased Erk-1/2 MAPK and Akt phosphorylation, which were detectable at the earliest time point examined (5 minutes). SU11248 inhibited Erk-1/2 MAPK and Akt activation at all time points examined.
We next determined the effect of SU11248 on PDGF-stimulated LX-2 viability. Cells were supplemented with PDGF-BB (20 ng/mL) in the presence or absence of SU11248 for 48 hours. Cell viability was then determined by measuring MTT reduction. As shown in Fig. 7F, incubation with 20 ng/mL PDGF-BB resulted in a significant increase in LX-2 viability compared with the control condition. In contrast, LX-2 cell viability was reduced significantly after 48 hours of incubation with SU11248. These results suggest that the inhibitory effect of SU11248 on PDGFR activity may contribute to the antifibrotic effect observed in cirrhotic livers after SU11248 treatment.
This study revealed that cirrhosis is accompanied not only by splanchnic vessel growth but also by extensive hepatic angiogenesis. Additionally, newly formed hepatic vasculature expressed VCAM-1 and ICAM-1. The nature of the inflammatory response is regulated by the induction of adhesion molecules in endothelial cells, which is a necessary event in the recruitment of inflammatory cells to sites of inflammation. Among these adhesion molecules, VCAM-1 and ICAM-1 appear to play a major role in the adhesion and transmigration of macrophages and lymphocytes across endothelial cells in a variety of acute and chronic inflammatory diseases. 19, 20 Double immunohistochemical staining demonstrated that VCAM-1 and ICAM-1 expression in the hepatic vasculature was adjacent to areas of local inflammatory infiltrate. Interestingly, hepatic vascular density as well as VCAM-1 and ICAM-1 endothelial expression were higher as cirrhosis progressed. In this scenario, we hypothesized that liver endothelial cells are activated to form new vessels and to express adhesion molecules. Circulating cells are then attracted to the activated vasculature, where they adhere to and migrate into the liver parenchyma.
VEGF-A and the angiopoietin families of growth factors are essential for vascular growth. 21, 22 We found a significant correlation between placental growth factor, VEGF-A, ang-1, and ang-2 expression and vascular density during cirrhosis progression. Several studies have shown that these angiogenic factors are up-regulated by cytokines and chemokines that are released by leukocytes and damaged hepatocytes. 23 These observations suggest that initially, proinflammatory cytokines may contribute to the up-regulation of these growth factors. Because tissue hypoxia often occurs in inflamed tissue after extensive fibrosis, 24 the colocalization of VEGF-A and ang-2 with hypoxic areas was assessed. Our findings showed a clear colocalization between VEGF-A/ang-2 overexpression and hypoxia, in agreement with earlier studies. 25
Linkage of angiogenesis to inflammatory infiltrate in cirrhotic livers suggests that angiogenesis inhibitors may interfere with progression of the disease. In fact, studies in experimental models of cirrhosis have shown that angiogenic inhibitors such as TNP-470 and neutralizing monoclonal antibodies anti-VEGFR1 or anti-VEGFR2 can effectively decrease liver fibrosis. 26, 27 Consistent with these reports, we have also demonstrated that cirrhotic rats treated with SU11248 showed a significant decrease in hepatic fibrosis. However, our study extends these findings in several significant ways. First, SU11248 treatment decreases inflammatory infiltrate in cirrhotic livers (Fig. 6). This beneficial effect of SU11248 is likely due to the decrease in the number of hepatic vessels expressing VCAM-1 and ICAM-1. Second, SU11248 treatment significantly decreased portal pressure in cirrhotic rats (Fig. 7D), which may be ascribed to several mechanisms. First, considerable evidence has demonstrated that HSCs are the primary source of extracellular matrix accumulation in livers, and PDGFR signaling is known to have a major role in this process. Therefore, it is likely that SU11248 decreased α-SMA and extracellular matrix accumulation in cirrhotic livers through the inhibition of the PDGF signaling pathway in HSCs. This hypothesis is supported by the results obtained with the LX-2 cell line where we demonstrated that the PDGF-BB–dependent increase in cell viability was reduced significantly after SU11248 treatment (Fig. 7F).
The increase in portal blood flow is another important component contributing to portal hypertension. There is significant evidence suggesting that the increase in portal blood flow is due not only to splanchnic vasodilation but also to an enlargement of the splanchnic vascular tree caused by angiogenesis. 7, 28, 29 Therefore, we anticipated that the significant inhibition of angiogenesis promoted by SU11248 in this anatomic region might translate into a significant decrease in portal blood flow.
In conclusion, this study demonstrates that therapies with several molecular targets against angiogenesis, inflammation, and fibrosis might be beneficial in the treatment of cirrhosis. The advantage of using multitargeted inhibitors such as SU11248 is further supported by the observation that SU5416, which is similar to SU11248 but exclusively targets VEGFRs, failed to modify portal pressure in the experimental model of partial portal vein ligation. 30 This discrepancy may be explained by the lack of specificity of SU5416 for targeting PDGFR compared with the multitargeting capability of SU11248.