Administration Of Biliverdin Research Articles

Biliverdin-induced brainstem auditory evoked potential abnormalities in the jaundiced Gunn rat

Brainstem auditory evoked potential (BAEP) abnormalities occur in jaundiced Gunn rats given sulfadimethoxine to displace bilirubin bound to serum albumin, releasing it into the tissues. One problem with the model is that after displacement, plasma bilirubin levels drop and do not correlate with neurological dysfunction. In this report, we administered biliverdin, the immediate precursor of bilirubin, in 15- to 17-day-old Gunn rat pups to create an improved model of bilirubin-induced neurological dysfunction. Total plasma bilirubin (TB) levels were measured with a Leica bilirubinometer. Biliverdin (40 mg/kg) or phosphate-buffered saline (PBS) was administered either once and BAEPs recorded 8 h later or twice, 12 h apart, and BAEPs recorded 24 h after the initial injection. A single biliverdin injection produced a significantly decreased amplitude of BAEP wave III, 1.21 ± 0.25 vs. 0.49 ± 0.27 μV (control vs. biliverdin). The two-injection paradigm resulted in a significantly elevated TB (9.9 ± 1.2 vs. 14.9 ± 3.1 mg/dl; control vs. biliverdin), significant increases in I–II (1.15 ± 0.08 vs. 1.42 ± 0.09 ms) and I–III (2.17 ± 0.08 vs. 2.5 ± 0.13 ms) interwave intervals and a decrease in the amplitude of wave III (1.36 ± 0.30 vs. 0.38 ± 0.26 μV). Additionally, there were significant correlations between TB and the amplitude of wave III ( r 2 = 0.74) and TB and the I–III interwave interval ( r 2 = 0.51). In summary, biliverdin administration in jaundiced Gunn rat pups produces BAEP abnormalities consistent with those observed in the sulfadimethoxine model and human newborn hyperbilirubinemia and resulted in increased plasma bilirubin levels that correlate with the degree of neurological dysfunction.

Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes

Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF 2α and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF 2α, P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 ± 0.8 cells/ml blood in control rats vs. 48 ± 4.8 cells/ml blood in diabetic rats ( P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 ± 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 ± 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 ± 2.3 ( P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 ± 2.3 to 29 ± 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes.

Products of heme oxygenase and their potential therapeutic applications

Heme oxygenase 1 (HO-1) is induced in response to cellular stress and is responsible for converting the prooxidant heme molecule into equimolar quantities of biliverdin (BV), carbon monoxide (CO), and iron. BV is then converted to bilirubin (BR) by the enzyme biliverdin reductase. Experimental evidence suggests that induction of the HO system is an important endogenous mechanism for cytoprotection and that the downstream products of heme degradation, CO, BR, and BV, may mediate these powerful beneficial effects. These molecules, which were once considered to be toxic metabolic waste products, have recently been shown to have dose-dependent vasodilatory, antioxidant, and anti-inflammatory properties that are particularly desirable for tissue protection during organ transplantation. In fact, recent work has demonstrated that administration of exogenous CO, BR, or BV may offer a simple, inexpensive method to substitute for the cytoprotective effects of HO-1 in a variety of clinically applicable models. This review will attempt to summarize the relevant biochemical and cytoprotective properties of CO, BR, and BV, and will discuss emerging studies involving the therapeutic applications of these molecules in the kidney and other organ systems.

Biliverdin administration protects against endotoxin-induced acute lung injury in rats

Given the high morbidity and mortality rates associated with pulmonary inflammation in sepsis, there is a pressing need for new therapeutic modalities to prevent acute respiratory distress. The enzyme heme oxygenase-1 (HO-1) provides potent cytoprotection against lung injury; however, the mechanism by which it does so is unclear. HO-1 catabolizes heme into biliverdin (BV), which is rapidly converted to bilirubin by BV reductase. We tested the hypothesis that BV administration could substitute for the effects observed with HO-1. Using the well-described rat model of LPS-induced shock, we demonstrate that exposure to BV imparts a potent defense against lethal endotoxemia systemically, as well as in the lungs, and effectively abrogates the inflammatory response. BV administration before a lethal dose of LPS leads to a significant improvement in long-term survival: 87% vs. 20% in sham-treated controls. BV treatment suppressed LPS-induced increases in lung permeability and lung alveolitis and significantly reduced serum levels of the LPS-induced proinflammatory cytokine IL-6. Moreover, bilirubin administered just after LPS also abrogated lung inflammation. BV treatment also augmented expression of the anti-inflammatory cytokine IL-10. Similar effects on production were observed with BV treatment in vitro in mouse lung endothelial cells and RAW 264.7 macrophages treated with LPS. In conclusion, these data demonstrate that BV can modulate the inflammatory response and suppress pathophysiological changes in the lung and may therefore have therapeutic application in inflammatory disease states of the lung.

Biliverdin Administration Prevents the Formation of Intimal Hyperplasia Induced by Vascular Injury

Autologous vein grafts and balloon angioplasty are still commonly used for arterial reconstructive procedures. Their success is limited by the development of intimal hyperplasia (IH). Biliverdin (BVD), one of the by-products of heme degradation, has been shown to have potent antioxidant and antiinflammatory effects. We hypothesized that BVD administration would protect vascular tissue against vascular injury. The effects of BVD administration against IH after vascular injury were analyzed in an arterialized vein graft model and a balloon injury model in rats. BVD treatment significantly suppressed the development of IH in both models compared with those without BVD. The mechanisms by which BVD treatment inhibits IH development might include decreasing c-Jun NH2 terminal kinase activation and preventing apoptosis of endothelial cells. BVD also suppressed vascular smooth muscle cell migration in vitro. BVD administration prevented IH associated with arterialized vein graft vasculopathy or balloon angioplasty-induced vessel injury. These results suggest that a treatment regimen with exogenous BVD administration could provide an effective therapeutic adjunct to facilitate transfer of experimental treatments for vascular injury to the clinic.

Heme Oxygenase-1-Generated Biliverdin Ameliorates Experimental Murine Colitis

Heme oxygenase-1 (HO-1) seems to have an important protective role in acute and chronic inflammation. The products of heme catalysis, biliverdin/bilirubin, carbon monoxide (CO), and iron (that induces apoferritin) mediate the beneficial effects of HO-1. Blockade of HO-1 activity results in exacerbation of experimental colitis. We tested whether HO-1 has protective effects in the development of colitis and determined that specific enzymatic products of HO-1 are responsible for these effects. Colitis was induced by oral administration of dextran sodium sulfate (5%) to C57BL/6 mice for 7 days. HO-1 was up-regulated by cobalt-protoporphyrin (5 mg/kg, intraperitoneally). Biliverdin, exogenous CO, or the iron chelator desferrioxamine was administered to other groups. Cobalt-protoporphyrin treatment resulted in significant up-regulation of HO-1 protein in mucosal and submucosal cells. Induction of HO-1 was associated with significantly less loss of body weight in mice with induced colitis (-12% versus -22% in the control animals, P < 0.001). Development of diarrhea and gastrointestinal hemorrhage was substantially delayed in animals in which HO-1 was induced, and mucosal injury was significantly attenuated. Administration of CO or desferrioxamine alone had no significant effects, whereas enhanced protection with lesser evidence of bowel inflammation was observed with systemic biliverdin administration (50 micromol/kg, 3 times per day, intraperitoneally). We conclude that heightened HO-1 expression or administration of biliverdin ameliorates dextran sodium sulfate-induced experimental colitis. Novel therapeutic strategies based on HO-1 and/or biliverdin administration may have use in inflammatory bowel disease.

Biliverdin protects against acute Tnf-α-induced fulminant hepatitis in vivo and hepatocyte cell death in vitro

Background. Hepatic failure can occur as a consequence of multiple disease processes. These disease processes also involve the upregulation of “protective genes” such as the stress-inducible enzyme heme oxygenase-1 (HO-1). HO-1 is the rate-limiting enzyme in the breakdown of heme into biliverdin, iron, and carbon monoxide. Biliverdin gets reduced by biliverdin reductase (BVR) to bilirubin, which is subsequently oxidized back into biliverdin. This sets up a powerful antioxidant cycle and thus biliverdin can serve to protect against oxidative stress. We hypothesized that biliverdin administration to mice prevent TNF-α-induced hepatitis in vivo and protects against hepatocyte apoptosis in vitro. Methods. Mice were treated with TNF-α/D-galactosamine (0.3 μg/8 mg/mouse, i.p., respectively) to induce hepatitis. Groups of mice were pretreated 16 h prior to TNF/D-Gal with biliverdin (5 mg/kg, i.p), or vehicle control. Mice were sacrificed 6 h later and serum alanine aminotransferase (ALT) was measured. Primary mouse and human hepatocytes were treated with TNF-α (10 ng/ml) and Actinomycin-D (200 ng/ml) to induce hepatocyte cell death. One group of hepatocytes was pretreated with biliverdin (0–500 μM) 16 h prior to the addition of TNF/ActD. Another group of biliverdin-treated human hepatocytes was pretreated 36 h with either an adenovirus containing an siRNA against human BVR or a control adenovirus (ψ5; 30 MOI). Hepatocyte viability was then assessed 12 h later via crystal violet staining. Results. TNF/D-Gal treatment of mice resulted in elevated serum ALT values (600 ± 200 IU/ml). Treatment with biliverdin resulted in significantly reduced ALT values of 23 ± 8 IU/ml ( P < 0.01). In vitro, biliverdin pretreatment significantly protected against TNF-induced cell death in a dose-dependent manner. TNF/ActD decreased hepatocyte viability to 34.0% in rat and 59% in human cell cultures. Biliverdin (250 μM) pretreatment increased viability to 68 and 78% in rat and human hepatocytes, respectively ( P < 0.05). Additionally, in human cells, the administration of Ad-BVR siRNA attenuated the protective effects of biliverdin treatment (viability of 60.0%), whereas biliverdin-treated cultures infected with control adenovirus continued to show increased survival (viability of 82.2%). Conclusions. Biliverdin protects against acute fulminant hepatitis and hepatocyte death. This appears to be dependent on BVR expression. Biliverdin/BVR may prove to be useful clinical adjuncts in the treatment of liver injury.

Heme oxygenase exerts a protective role against oxidative stress in soybean leaves

We have previously demonstrated that the induction of heme oxygenase-1 (EC 1.14.99.3) plays a protective role for mammalian cells against oxidative stress. Here, we investigated for the first time the possible role of heme oxygenase-1 as an antioxidant defense in leaves of soybean plants. Treatment with 200μM Cd during 48h caused a 70% increase in thiobarbituric acid reactive substances, whereas GSH decreased 67%, guaiacol peroxidase and total superoxide dismutase also inhibited 49% and 46%, respectively. Two hundred micromolar of Cd produced the overexpression of heme oxygenase-1, as well as a 4.5-fold enhancement of its activity. Administration of biliverdin partially prevented the effects caused by Cd. Pretreatment with Zn protoporphyrin IX, a potent inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given before Cd, it completely prevented the enzyme induction increasing the levels of oxidative stress parameters. Collectively, these results indicated that although plant heme oxygenases share little homology to heme oxygenases from non-plant species, they also play an important protective role against oxidative cell damage.

Biliverdin protects the functional integrity of a transplanted syngeneic small bowel

Background & Aims: Heme oxygenase-1 (HO-1) protects against inflammation in many disease models. By degrading heme, HO-1 generates carbon monoxide (CO), iron and biliverdin. We investigated whether biliverdin would protect rat syngeneic small intestinal transplants (SITx) against damage and, if so, by what mechanism Methods: Motility was assessed by organ bath techniques. Inflammatory cytokines and mediators were assessed by RT-PCR and spectrophotometric assays. Myeloperoxidase histochemistry for neutrophils was performed in jejunal segments. Western blots were performed for biliverdin reductase and HO-1 expression. Permeability was expressed as the mucosal to serosal clearance of fluorescent dextran in everted gut sacs. NF-κB activation was assessed via EMSA. Results: Biliverdin significantly improved survival of recipients following SITx after prolonged intestinal ischemia (6 hours). Biliverdin treatment (1) led to a significant decrease in mRNA expression of iNOS, Cox-2, and ICAM-1 as well as the inflammatory cytokines IL-6 and IL-1β; (2) decreased neutrophil infiltration into the jejunal muscularis; and (3) prevented SITx-induced suppression of intestinal circular muscle contractility. Conclusions: Biliverdin administration attenuates transplantation-induced injuries to the small bowel by its anti-inflammatory action. Importantly, biliverdin enhanced recipient survival. A comparison of the mechanisms by which biliverdin exerted these salutary effects compared with inhalation of CO, which we previously showed had salutary effects, suggests that the 2 compounds (biliverdin and CO) exert their effects in part by different mechanisms. This implies that the different products of HO-1 action on heme may exert protective effects that are additive or synergistic.

BILIRUBIN SUPPRESSES NEOINTIMA FORMATION BY INTERACTING WITH THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN PATHWAY

O386* Aims: Induction of heme oxygenase 1 (HO-1) ameliorates the development of chronic allograft arteriosclerosis, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent antioxidants that play a role in maintenance of homeostasis. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. We used balloon injury (BI) as a model of the neointimal proliferation seen in transplant arteriosclerosis. Methods: BI was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats and congenic wild-type Wistar rats. The artery was harvested two weeks after BI, and neointima formation was assessed by quantifying intima/media ratio and luminal cross-sectional area narrowing (LCAN). In vitro, proliferation of primary vascular smooth muscle cells (VSMC) was measured by 3H-thymidine incorporation in the presence or absence of bilirubin/ biliverdin. Cell cycle progression was monitored by DNA content analysis; apoptosis by the Annexin V binding assay. Protein levels were detected by western blot. Immuncytochemistry was performed using antibody directed against the retinoblastoma tumor suppressor protein (pRb). Results: BI mediated neointima formation was significantly reduced in hyperbilirubinemic Gunn rats (serum bilirubin levels: 12.0 ± 2.5 mg/dl) as compared to control Wistar rats (LCAN 0.18 vs. 0.44; p < 0.05). Systemic administration of biliverdin, the precursor of bilirubin, to normal rats also suppressed neointima proliferation (LCAN 0.29 vs. 0.43; p < 0.05). In vitro, bilirubin inhibited VSMC proliferation in a dose-dependent manner. Bilirubin (200 μM) arrested VSMC cell cycle progression at the G0/G1 phase, while it did not increase apoptosis (p=0.2816). Inhibition of cell cycle progression was mediated by reduced phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase 1/2 (JNK 1/2), down-regulation of cdk2, cyclins D1, E and A, and the resulting decreased phosphorylation of pRb. In nuclear extracts, lower total levels of pRb were found in bilirubin treated VSMCs, consistent with the findings that a significant portion of pRb was kept in the cytosol after bilirubin treatment as assessed by immunocytochemistry. Further, bilirubin treatment reduced expression levels of the transcription factor YY1, a zinc finger DNA-binding transcription factor known to regulate the expression of genes with important functions during cell growth and differentiation. The effects of bilirubin were independent of HO-1 expression, as demonstrated in HO-1−/− VSMCs. Conclusions: Biliverdin and bilirubin suppress both BI induced neointima formation in vivo and VSMC proliferation in vitro. Our data suggest that bilirubin treatment inhibits VSMC growth by impaired activation of p38 and JNK 1/2, resulting in two events. Firstly, the inhibition of pRb phosphorylation impedes the release of transcription factors such as YY1 that are important for VSMC growth. Secondly, there is reduced expression of the transcription factor YY1 itself, resulting in a suppressed capacity to induce DNA synthesis and transcriptional activity. Thus, administration of the bile pigments bilirubin and biliverdin may have a beneficial effect on VSMC proliferation in chronic allograft arteriopathy and may become a potent strategy for the treatment of chronic rejection.

Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts.

Biliverdin, a product of heme oxygenase-1 (HO-1) enzymatic action, is converted into bilirubin, which has been considered a waste product in the past. We now show that administration of biliverdin has a salutary effect in organ transplantation. A brief course of treatment with biliverdin leads to long-term survival of H-2 incompatible heart allografts. Furthermore, those recipients harboring long-surviving (>100 days) allografts were tolerant to donor antigens indicated by the acceptance of second donor strain hearts but not third-party grafts. Treatment with biliverdin decreased intragraft leukocyte infiltration and inhibited T cell proliferation. Likely related to tolerance induction, biliverdin interferes with T cell signaling by inhibiting activation of nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB), two transcription factors involved in interleukin-2 (IL-2) transcription and T cell proliferation, as well as suppressing Th1 interferon-gamma (IFN-gamma) production in vitro. These findings support the potential use of biliverdin, a natural product, in transplantation and other T cell mediated immune disorders.

Adenovirus-mediated heme oxygenase-1 gene expression stimulates apoptosis in vascular smooth muscle cells.

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme into biliverdin, iron, and carbon monoxide (CO). Although HO-1 is induced in vascular smooth muscle cells (SMCs), the biological role of HO-1 in these cells has not been completely characterized. In the present study, we overexpressed HO-1 in rat aortic SMCs by generating a recombinant defective adenovirus containing the rat HO-1 gene (AdHO-1) and examined the effect on SMC proliferation. Infection of SMCs with AdHO-1 resulted in a dose-dependent increase in the expression of HO-1 mRNA, protein, and activity. Infection of SMCs with AdHO-1 inhibited serum-stimulated SMC proliferation in a dose-dependent manner. In contrast, the control adenovirus expressing the green fluorescent protein failed to induce HO-1 expression and had minimal effects on SMC growth. Infection with AdHO-1 stimulated SMC apoptosis in a dose-dependent fashion, as demonstrated by DNA fragmentation, positive annexin V labeling, and caspase-3 activation. HO-1-mediated apoptosis was associated with a marked increase in the expression of the proapoptotic protein p53. Finally, the exogenous administration of biliverdin and bilirubin stimulated SMC apoptosis. In contrast, the administration of CO or iron failed to induce cell death. These results demonstrate that overexpression of HO-1 or the exogenous administration of biliverdin or bilirubin stimulates SMC apoptosis. Adenovirus-mediated transfer of the HO-1 gene may provide a novel therapeutic approach in treating occlusive vascular disease.

A beneficial role of bile pigments as an endogenous tissue protector: Anti-complement effects of biliverdin and conjugated bilirubin

Bile pigments possess and anti-complement property and could be involved in tissue protection. In this study, we examined the physiological actions of bile pigments, which had been generally regarded as waste catabolites. Biliverdin inhibited complement cascade reaction in vitro, especially at the C1 step in the classical pathway at low micromolar concentrations. Further, Forssman anaphylaxis in guinea pigs, being closely associated with complement reactions, was inhibited by oral or intravenous administration of biliverdin. Conjugated bilirubin also showed an inhibitory effect on complement-dependent reactions in vitro. From These observation, we propose a hypothesis that the pigments serves as endogenous tissue protectors by multiple lines of mechanisms including antioxidant and annti-complement actions.

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea).

Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of 3H-biliverdin were detected in hepatic bile following the intravenous administration of 3H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.

Induction of ornithine decarboxylase by biliverdin in rat liver

The administration of biliverdin (0.1mg/g of body weight) into the peritoneal cavity of rats resulted in the induction of ornithine decarboxylase in the liver. When the temporal relationships between the changes in intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level, cyclic AMP-dependent protein kinase activity and the induction of ornithine decarboxylase were investigated, the concentration of cyclic AMP increased significantly 2 h after the administration of biliverdin, while cyclic AMP-dependent protein kinase was activated after 2–4 h. The hepatic ornithine decarboxylase activity began to increase 4 h after biliverdin injection. These results suggest that there is some sequential relationship between the increase of cyclic AMP, the activation of cyclic AMP-dependent protein kinase and the induction of ornithine decarboxylase although the direct correlation of these three events remains to be elucidated.

皮下組織内胆汁色素生成過程に関する組織顕微分光学的研究

The formation of bilirubin with the absorption maximum at 478-480 mμ from verdohemoglobin could be scarecely decided, because the mixture of decompostion from the oxyhemoglobin produced by the subcutaneous injection of verdohemoglobin to white rat.2. The appearance of bilirubin was observed at the relatively early period on the administration of biliverdin to white rat and the apperance of relatively numerous hematodin crystal was observed.3. On the subcutaneous administration of hematoidin to white rat, it elapsed the product displaying the absorption maximum at 630 mμ and resolved to bilirubin without the formation of methemalbumin.