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(The FASEB Journal. 2007;21:81-87.)
© 2007 FASEB

Ribavirin inhibits angiogenesis by tetrahydrobiopterin depletion

Martin Michaelis*, Ruth Michaelis{dagger}, Tatyana Suhan*, Helmut Schmidt{ddagger}, Annisuddin Mohamed{dagger}, Hans Wilhelm Doerr* and Jindrich Cinatl, Jr.*,1

* Institut für Medizinische Virologie, Klinikum der J.W. Goethe Universität, Paul Ehrlich-Str. 40, Frankfurt am Main, Germany;

{dagger} Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe Universität, Theodor-Stern-Kai-7, Frankfurt am Main, Germany;

{ddagger} Pharmazentrum Frankfurt, Institut für Klinische Pharmakologie, Klinikum der J.W. Goethe Universität, Theodor-Stern-Kai-7, Frankfurt am Main, Germany

1Correspondence: Institut für Medizinische Virologie, Klinikum der Johann Wolfgang Goethe-Universität, Paul Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany. E-mail: cinatl{at}em.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribavirin is a broad-spectrum antiviral drug that is used to treat hepatitis C virus (HCV)-infected patients. The virological response after ribavirin treatment appears to be insufficient to fully explain ribavirin-induced beneficial effects. Angiogenesis plays a pathogenic role in HCV-induced liver damage. Here, we investigated the influence of therapeutic ribavirin concentrations on angiogenesis. Ribavirin inhibited endothelial cell tube formation in vitro and vessel formation in the chick chorioallantoic membrane assay in vivo. Ribavirin inhibits inosine monophosphate dehydrogenase, which causes depletion of cellular GTP and in turn reduction of cellular tetrahydrobiopterin levels. The availability of tetrahydrobiopterin limits NO production by endothelial NO synthase. Ribavirin reduced levels of tetrahydrobiopterin (as revealed by HPLC), NO (as revealed by electron spin resonance spectroscopy), and cGMP (as revealed by RIA) in endothelial cells. Addition of tetrahydrobiopterin or NO prevented ribavirin-induced tube formation inhibition. In conclusion, angiogenesis inhibition by ribavirin has not been described before. This inhibition may contribute to ribavirin-induced pharmacological effects including adverse events.—Michaelis, M., Michaelis, R., Suhan, T., Schmidt, H., Mohamed, A., Doerr, H. W., Cinatl, Jr., J. Ribavirin inhibits angiogenesis by tetrahydrobiopterin depletion.


Key Words: endothelial cell • hepatitis C • inosine monophospate dehydrogenase • NO


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RIBAVIRIN HAS ANTIVIRAL ACTIVITY against a broad range of DNA and RNA viruses (1) . Pegylated IFN {alpha} (PEG-IFN {alpha}) in combination with the ribavirin is currently the standard treatment for chronic hepatitis C virus (HCV) infection (2 , 3) .

Different hypotheses have been raised how ribavirin might inhibit HCV replication: 1) inhibition of inosine monophosphate (IMP) dehydrogenase that leads in turn to depletion of intracellular GTP pools, 2) moderate direct inhibition of HCV RNA-dependent RNA polymerase, and/or 3) induction of error catastrophe resulting from the accumulation of (lethal) mutations in the viral genome (3) . Moreover, ribavirin has been suggested to induce a favorable shift of the immune response from T helper cells 2 (TH2) to TH1 (3 4 5) . All of these mechanisms may contribute to the anti-HCV effects of ribavirin. However, ribavirin monotherapy does not exert significant effects on virological response (5 6 7) . Nevertheless, ribavirin improved transaminases transiently and had a significant beneficial effect on liver histology. These effects seem to be mediated via mechanisms unrelated to virological response and may delay cirrhosis development (6 , 7) .

Angiogenesis has been reported to play a significant pathogenic role in liver damage in chronic hepatitis C patients (8 9 10 11) . Clinical and experimental data suggest a link between increased angiogenesis and liver fibrosis as well as between increased angiogenesis and formation/progression of HCV-induced hepatocellular carcinoma (12 13 14 15 16) . The angiogenesis inhibitor angiostatin inhibited experimental liver fibrosis in mice (14) . In addition, angiogenesis inhibitors are discussed as potential treatment options for hepatocellular carcinoma (15) .

Inhibition of IMP dehydrogenase by ribavirin leads to depletion of cellular GTP and in turn to decreased cellular tetrahydrobiopterin levels (17) . The availability of tetrahydrobiopterin limits the NO production by endothelial NO synthase (eNOS) (18) , and NO production by eNOS plays a critical role during angiogenesis (19) . Therefore, ribavirin may interfere with this central angiogenesis signaling pathway.

Here, we investigated the influence of ribavirin on angiogenesis as indicated by endothelial cell proliferation and endothelial cell formation of tube-like structures. The antiangiogenic effects of ribavirin were confirmed in vivo in the chick chorioallantoic membrane (CAM) assay.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ribavirin (Virazole®) was obtained from Valeant Pharmaceuticals Germany GmbH (Eschborn, Germany). DETA NONOate and tetrahydrobiopterin were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Cells
Human umbilical vein endothelial cells (HUVEC) were cultivated as described (20) . Cells were seeded onto Matrigel (BD Biosciences, Heidelberg, Germany; diluted 1:80 in culture medium)-coated culture flasks and grown in Iscove’s modified Dulbecco’s medium supplemented with 15% fetal calf serum (FCS), 5% pooled human serum (Blood Bank of The German Red Cross, Frankfurt am Main, Germany), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 ng/ml basic fibroblast growth factor (bFGF).

Immunoblotting
Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE, as described (21) . Proteins were detected using specific antibodies against ß-actin (Sigma, Taufkirchen, Germany) or eNOS (Cell signaling, Beverly, MA, USA) and were visualized by enhanced chemiluminescence (ECL) using a commercially available kit (Amersham, Germany).

Viability assay
Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (22) modified as described before (20) . In brief, HUVEC (103 cells/ml) were seeded onto 96-well microtiter plates in serum containing serial dilutions of ribavirin. After 5 d of incubation, MTT (1 mg/ml) was added, and after an additional 4 h cells were lysed in a buffer containing 20% (w/v) SDS and 50% N,N-dimethylformamide adjusted to pH 4.5. Absorbance at 570 nm was determined for each well using a 96-well multiscanner. After subtracting background absorbance, results are expressed as cell number compared to control cells that were maintained in the presence of solvent.

Cell cycle analysis
Cell cycle was determined using a commercial kit (BD Biosciences) following the manufacturer’s instructions as described previously (21) .

In vitro tube formation assay
Endothelial cell tube formation was assessed as described (20) . Briefly, 96-well plates were coated with cold Matrigel (50 µl/well), which was allowed to polymerize at room temperature for ~30 min. Thereafter, 100 µl of a suspension of HUVEC (5x104 cells/ml) was seeded onto the Matrigel and cultured overnight in IMDM, supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5% (v/v) FCS. Tube formation was assessed after 12 h and quantified by determining the mean number of branching points in at least three different wells.

Chick chorioallantoic membrane assay
All experiments with chick embryos were carried out in ovo as described (20) . Briefly, methylcellulose discs with or without ribavirin were placed onto the 8-d-old CAMs. Two to three discs were placed on each CAM ~10 mm apart. Evaluation of the CAMs was performed 4 d after the application of the disc. To better visualize the vascular system of the CAM, 20% Luconyl Black (BASF, Ludwigshafen, Germany) in PBS was injected into a vitelline vein using glass capillaries.

Measurement of cellular tetrahydrobiopterin levels
Tetrahydrobiopterin levels were measured as described by Fukushima and Nixon (23) . Homogenized cells were oxidized at acidic pH with iodine and extracted by solid-phase extraction. Concentrations of total biopterin were determined by HPLC (Gemini C18 column, Phenomenex, Aschaffenburg, Germany) coupled to mass spectrometry (4000 Q TRAP triple quadrupole mass spectrometer with a Turbo V source ion spray, Applied Biosystems, Darmstadt, Germany).

Measurement of NO levels
NO production was detected in washed human platelets by electron spin resonance spectroscopy (ESR) by measuring the rate of formation of paramagnetic mono-nitrosyl-iron complex (MNIC), as described (24) . The ESR spectra were recorded at 77 K on a BRUKER ESR 300E at a frequency of 9.47 GHz, modulation frequency 100 kHz, modulation amplitude 0.5 mT, microwave power 20 mW, and time constant 0.1–1.3 s.

Measurement of cellular cyclic guanosine monophosphat (cGMP) levels
cGMP levels were determined in isobutyl methylxanthine (IBMX, 3 µmol/L)-treated endothelial cells using a specific RIA (PerkinElmer, Zaventem, Belgium) as described before (24) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ribavirin inhibits endothelial cell proliferation
Endothelial cell proliferation is an initial step in the angiogenic process. Trypan blue assay revealed that ribavirin did not exert cytotoxic effects in HUVEC in concentrations up to 100 µg/ml. The relative amount of dead (trypan blue-stained) cells was <5% in ribavirin-treated as well as in nontreated control cells. The MTT assay showed no significant antiproliferative effects in ribavirin concentrations ≤20 µg/ml after five days (Fig. 1 A). A G1/S cell cycle block could be detected in HUVEC treated with ribavirin 100 µg/ml for 48 h but not in HUVEC treated with ribavirin 20 µg/ml or 2 µg/ml (Fig. 1B ).


Figure 1
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  		Figure 1. Influence of ribavirin on endothelial cell proliferation and cell cycle. A) Concentration-dependent influence of ribavirin on endothelial cell viability as determined by MTT assay after 5 d incubation. The decreased cell viability is the result of cell growth inhibition, since no cytotoxicity was detected in the trypan blue assay. B) Cell cycle analysis of endothelial cells treated for 48 h without or with ribavirin. *P < 0.05 relative to control.

Ribavirin inhibits endothelial cell tube formation in vitro and angiogenesis in the chick chorioallantoic membrane (CAM) assay in vivo
Angiogenesis involves the organization of endothelial cells into a network of tube-like structures. Ribavirin (20 µg/ml) preincubation inhibited endothelial cell tube formation in a time-dependent manner. Longer preincubation periods resulted in increased tube formation inhibition (Fig. 2 A). Pretreatment was necessary since HUVEC incubation with ribavirin (20 µg/ml) during cultivation on Matrigel did not affect tube formation (data not shown). Five-day pretreatment with ribavirin concentrations between 2.5 µg/ml and 20 µg/ml inhibited HUVEC tube formation in a concentration-dependent manner (Fig. 2B, C ). Ribavirin concentrations ≤1 µg/ml did not significantly inhibit tube formation (data not shown).


Figure 2
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  		Figure 2. Influence of ribavirin on endothelial cell tube formation and vessel formation in the chick chorioallantoic membrane (CAM). A) Endothelial cell tube formation after 24, 72, or 120 h pretreatment with ribavirin 20 µg/ml relative to nontreated control. B) Representative photographs showing endothelial cell tube formation of control cells or of cells pretreated with ribavirin for five days. C) Endothelial cell tube formation after 5 d pretreatment with ribavirin relative to nontreated control. D) Representative photographs showing vessel formation with or without ribavirin (20 µg/ml) treatment in the CAM. *P < 0.05 relative to control.

To assess vessel development in the CAM assay, methylcellulose discs containing either solvent (saline, 8 eggs) or ribavirin (20 µg/ml, 8 eggs) were applied to the CAM on day 8 of embryo development. After another four days, the CAMs underlying the saline-containing disc had matured normally while the CAMs underlying the ribavirin-containing disc were poorly vascularized (Fig. 2D ).

Ribavirin inhibits production of biopterin, NO, and cyclic GMP (cGMP) in endothelial cells
Ribavirin inhibits IMP dehydrogenase, which causes reduction of intracellular GTP pools and in turn reduction of cellular tetrahydrobiopterin levels (17) . The availability of tetrahydrobiopterin limits the NO production by eNOS (18) . NO activates soluble guanylyl cyclase and thus elicits an increase in intracellular levels of cGMP, which then further mediates proangiogenic effects in endothelial cells (19) . Ribavirin 20 µg/ml significantly inhibited the cellular production of tetrahydrobiopterin, NO, and cGMP (Fig. 3 ).


Figure 3
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  		Figure 3. Influence of ribavirin on cellular tetrahydrobiopterin, NO, and cGMP levels. Endothelial cells were treated with ribavirin 20 µg/ml for five days and cellular levels of (A) tetrahydrobiopterin, (B) NO, or (C) cGMP were determined relative to control. *P < 0.05 relative to control.

Ribavirin did not influence eNOS levels in HUVEC (data not shown).

Addition of tetrahydrobiopterin or exogenous NO prevents ribavirin-induced tube formation inhibition in endothelial cells
If reduction of cellular tetrahydrobiopterin levels and in turn cellular NO levels were the reason for ribavirin-induced tube formation inhibition in HUVEC, addition of tetrahydrobiopterin or NO should prevent tube formation inhibition. Tetrahydrobiopterin 10 µM was added to ribavirin 20 µg/ml treated HUVEC for 5 d. The NO-donor DETA NONOate (10 µM) was added daily during the 5-day ribavirin treatment period. Both treatments prevented ribavirin-induced tube formation inhibition (Fig. 4 ).


Figure 4
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  		Figure 4. Influence of tetrahydrobiopterin or NO on ribavirin-induced endothelial cell tube formation inhibition. Representative photographs (A) and quantitative analysis (B) of tube formation of endothelial cells treated with ribavirin (20 µg/ml 5 d), tetrahydrobiopterin (10 µM 5 d), the NO-donor DETA NONOate (10 µM added daily for 5 d), or with combinations of ribavirin and tetrahydrobiopterin or NO. *P < 0.05 relative to control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data presented here show that the IMP dehydrogenase inhibitor ribavirin prevents angiogenesis in vitro and in vivo. IMP dehydrogenase inhibitors cause depletion of cellular GTP and in turn decrease of cellular tetrahydrobiopterin levels (17) . The availability of tetrahydrobiopterin limits the NO production by eNOS (18) , and NO plays a critical role during angiogenesis (19) . NO activates soluble guanylyl cyclase and thus elicits an increase in intracellular levels of cGMP, which then further mediate proangiogenic effects in endothelial cells (19) . In accordance, our data show that ribavirin reduces the levels of tetrahydrobiopterin, NO, and cGMP in endothelial cells. Moreover, addition of tetrahydrobiopterin or NO prevents ribavirin-induced tube formation inhibition. This indicates that ribavirin-induced tetrahydrobiopterin depletion is responsible for ribavirin-induced antiangiogenic effects.

In rat microvascular endothelial cells, tetrahydrobiopterin had been demonstrated to regulate endothelial cell proliferation (25) . The IMP dehydrogenase inhibitor mycophenolic acid had been shown to inhibit angiogenesis in vitro and in vivo (26 , 27) . The investigated mycophenolic acid concentrations caused inhibition of endothelial cell proliferation as well as of endothelial cell tube formation (26) . In contrast, our results reveal that ribavirin can inhibit tube formation in concentrations below antiproliferative concentrations. Ribavirin 100 µg/ml inhibited endothelial cell proliferation, whereas concentrations ≤20 µg/ml did not. Tube formation inhibition was detected in ribavirin concentrations as low as 2.5 µg/ml. This finding suggests that the reduction of cellular tetrahydrobiopterin levels may cause antiangiogenic effects, although the extent of tetrahydrobiopterin depletion is insufficient to inhibit endothelial cell proliferation.

Clinically achievable ribavirin concentrations have been reported to be ≥2.5 µg/ml (28 29 30) . In patients with impaired renal function ribavirin plasma concentrations >100 µg/ml were detected (31) . Thus, antiangiogenic concentrations are in the range of therapeutic plasma levels. Antiangiogenic effects are insufficient to explain the ribavirin-induced reduced virologic relapse observed after IFN therapy (3) . However, ribavirin monotherapy improved transaminases transiently and had a significant beneficial effect on liver histology. These effects seem to be mediated via mechanisms unrelated to virological response and may delay cirrhosis development (6 , 7) . Given the pathogenic role of angiogenesis in liver damage in chronic hepatitis C patients (8 9 10 11) , ribavirin-induced antiangiogenic effects may play a part in those ribavirin-induced histological and biochemical responses in hepatitis C patients that appear to be unrelated to virological response. Interestingly, two recent case reports propagate the clinical investigation of thalidomide, a drug that has been proven to possess a strong antiangiogenic potential (32) , for hepatitis C patients (33 , 34) .

Several congeners of ribavirin have been investigated as antiviral drugs (35) . EICAR (5-Ethynyl-1-ß-D-ribofuranosylimidazole-4-carboxamide) has been shown to strongly inhibit IMP dehydrogenase and to exert a greater antiviral potency when compared to ribavirin (35 , 36) . Viramidine, a prodrug of ribavirin that is converted to ribavirin in the liver (37) , and levovirin, the L-enantiomer of ribavirin with decreased antiviral activity, show favorable toxicity profiles compared to ribavirin (38 39 40 41 42) . Based on our findings that ribavirin inhibits angiogenesis, it would be of interest to investigate those derivatives for antiangiogenic activity.

Although hemolytic anemia and pulmonary infiltrates represent the main adverse events under ribavirin therapy, ribavirin accounts for a plethora of additional toxic effects (43) . Therefore, ribavirin-induced angiogenesis inhibition may be involved in adverse events observed under ribavirin therapy such as teratogenic effects or worsening of cardiac disease (43) . The World Health Organization (WHO) has estimated about 170 million people infected with HCV (44) . About 2.7 million persons are estimated to be chronically HCV-infected in the United States (43) . Since PEG-IFN {alpha} plus ribavirin represents the standard medication for chronic hepatitis C patients (2 , 3) , ribavirin is a frequently used drug. The interference of ribavirin with a basal biological process like angiogenesis would be of general importance, not only for physicians and scientists engaged in infectious diseases, but for all that have to deal with ribavirin-treated patients.

In conclusion, our data show that therapeutic ribavirin concentrations inhibit angiogenesis in vitro. Moreover, ribavirin inhibits in vivo angiogenesis in the CAM. The mechanism of ribavirin-induced angiogenesis inhibition is depletion of cellular tetrahydrobiopterin and in turn inhibition of endothelial cellular NO production. Angiogenesis inhibition by ribavirin has not been described before. This inhibition may contribute to ribavirin-induced beneficial effects to hepatitis C patients that cannot be explained by virological response. In addition, ribavirin-induced angiogenesis inhibition may play a role for additional pharmacological and toxic effects, such as ribavirin-induced teratogenic effects or worsening of cardiac disease in ribavirin-treated patients.


   ACKNOWLEDGMENTS
 
This work was supported by the friendly society "Hilfe für krebskranke Kinder Frankfurt e.V." and its foundation "Frankfurter Stiftung für Krebskranke Kinder." The authors thank Sofia Aidanopoulou, Kerstin Euler and Janette Spitznagel for technical assistance.

Received for publication July 3, 2006. Accepted for publication August 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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