Mouse Model Of Erythropoietic Protoporphyria Research Articles

Bitopertin, a Selective Glycine Transporter 1 Inhibitor, Reduced Protoporphyrin IX (PPIX) Level and Improved Liver Fibrosis in a Mouse Model of Erythropoietic Protoporphyria (EPP)

Bitopertin, a Selective Glycine Transporter 1 Inhibitor, Reduced Protoporphyrin IX (PPIX) Level and Improved Liver Fibrosis in a Mouse Model of Erythropoietic Protoporphyria (EPP)

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

Liver metabolomics in a mouse model of erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a genetic disease that results from the defective mutation in the gene encoding ferrochelatase (FECH), the enzyme that converts protoporphyrin IX (PPIX) to heme. Liver injury and even liver failure can occur in EPP patients because of PPIX accumulation in the liver. The current study profiled the liver metabolome in an EPP mouse model caused by a Fech mutation (Fech-mut). As expected, we observed the accumulation of PPIX in the liver of Fech-mut mice. In addition, our metabolomic analysis revealed the accumulation of bile acids and ceramide (Cer) in the liver of Fech-mut mice. High levels of bile acids and Cer are toxic to the liver. Furthermore, we found that the major phosphatidylcholines (PC) in the liver and the ratio of total PC to PPIX in the bile were decreased in Fech-mut mice compared to wild type mice. A decrease of the ratio of PC to PPIX in the bile can potentiate the accumulation of PPIX in the liver because PC increases PPIX solubility and excretion. These metabolomic findings suggest that the accumulation of PPIX, together with the disruption of the homeostasis of bile acids, Cer, and PC, contributes to EPP-associated liver injury.

Protective action of antioxidants on hepatic damage induced by griseofulvin.

Erythropoietic protoporphyria (EPP) is a disease associated with ferrochelatase deficiency and characterized by the accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver, and skin. In some cases, a severe hepatic failure and cholestasis were observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in mice such as PROTO IX accumulation followed by cellular damage as wells as necrotic and inflammatory processes. The antioxidant defense system was also altered. The aim was to evaluate the possible protective effect of different antioxidant compounds: trolox (Tx), ascorbic acid (Asc), the combination Tx and Asc, melatonin (Mel), and the polyphenols: ellagic acid, quercetin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid on liver damage and oxidative stress markers in a mouse model of EPP. Coadministration of Gris with Tx, Asc, and its combination, or Mel mainly affected heme biosynthetic pathway, resulting in a decrease in ALA-S activity which was increased by Gris, while the tested polyphenols exerted a protective effect on oxidative stress, decreasing lipid peroxidation and the activity of some antioxidant enzymes. In conclusion, antioxidant compounds can only protect partially against the liver damage induced by Gris, reducing oxidative stress or acting on heme regulation.

Characterization of Mallory-Denk Bodies in a Mouse Model of Erythropoietic Protoporphyria

Characterization of Mallory-Denk Bodies in a Mouse Model of Erythropoietic Protoporphyria

Level of Expression of the Nonmutant Ferrochelatase Allele is a Determinant of Biochemical Phenotype in a Mouse Model of Erythropoietic Protoporphyria

Ferrochelatase (FECH) activity is decreased in erythropoietic protoporphyria (EPP), causing increased production and excretion of protoporphyrin. This study examined whether the level of expression of the nonmutant FECH allele is a determinant of phenotype in a mouse model of EPP that carries a heterozygous deletion of exon 10 in FECH. Two mice strains that had a two-fold difference in FECH mRNA levels in bone marrow and liver (low expressing C3H/HeJ and high expressing CBA/J) were used to establish congenic strains containing the mutation. Erythrocyte protoporphyrin levels in C3H/HeJ heterozygous mice were significantly higher than in their wildtype littermates, whereas levels in CBA/J heterozygous mice did not differ significantly from their wildtype littermates. Biliary excretion of protoporphyrin was also significantly higher in C3H/HeJ heterozygous mice. The levels of normal FECH mRNA in bone marrow measured by real time PCR were 138 +/- 30 copies per ug total RNA in C3H/HeJ +/- mice, 320 +/- 59 in C3H/HeJ +/+ mice and 634 +/- 38 in CBA/J +/+ mice. Levels in liver tissue of the mice differed significantly in the same pattern. Thus, the level of expression of the nonmutant FECH allele is a determinant of phenotype in a mouse model of EPP as has been demonstrated in human EPP.

Mdr1a/b nor Mdr2 P-glycoprotein is essential for biliary excretion of the hydrophobic heme precursor protoporphyrin ina griseofulvin-induced mouse model of erythropoietic protoporphyria: Torsten Plösch, Vincent W. Bloks, Juul F.w. Baller, Rick Havinga, Center for Liver, Digestive and Metabolic Diseases, Department of Pediatrics, University Hospital Groningen, Groningen Netherlands; Peter L.m. Jansen, Center for Liver, Digestive and Metabolic Diseases, Department of Internal

Mdr1a/b nor Mdr2 P-glycoprotein is essential for biliary excretion of the hydrophobic heme precursor protoporphyrin ina griseofulvin-induced mouse model of erythropoietic protoporphyria: Torsten Plösch, Vincent W. Bloks, Juul F.w. Baller, Rick Havinga, Center for Liver, Digestive and Metabolic Diseases, Department of Pediatrics, University Hospital Groningen, Groningen Netherlands; Peter L.m. Jansen, Center for Liver, Digestive and Metabolic Diseases, Department of Internal

Liver pathology and hepatocarcinogenesis in a long-term mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited disease of haem synthesis caused by a mutation in one of the alleles of the enzyme ferrochelatase. This mutation leads to partial deficiency of the enzyme, resulting in increased concentrations of protoporphyrin (PP) in blood, liver, and faeces. Five to ten per cent of patients with EPP develop severe liver disease characterized by the presence of PP deposits. This study used histochemistry and immunohistochemistry to investigate the histopathological features present in the livers of 44 mice with a heterozygous or homozygous point mutation in the ferrochelatase gene (fch/+ and fch/fch mice, respectively). Some fch/+ mouse livers showed mixed steatosis and large cell dysplasia. The livers of fch/fch mice showed periportal or septal fibrosis accompanied by an atypical ductular reaction. These findings suggest that the obstruction and damage of a proportion of large and small bile ducts by PP deposits cause an accumulation of PP in the parenchyma, which leads to damage and loss of hepatocytes due to the toxic effects of PP. The classical stages of hepatocarcinogenesis were observed and hepatic progenitor cells appear to be involved in this process. PP acts as the promoting agent and is probably also the initiating agent.

Mdr P-glycoproteins are not essential for biliary excretion of the hydrophobic heme precursor protoporphyrin in a griseofulvin-induced mouse model of erythropoietic protoporphyria

Hepatic complications in erythropoietic protoporphyria (EPP) have been attributed to toxic actions of accumulated protoporphyrin (PP). PP can only be removed via the bile but transport systems involved have not been defined. The aim of this study was to gain insight in the mode of biliary PP excretion, with emphasis on the potential contribution of the Mdr1 P-glycoprotein export pump and biliary lipids as PP carriers. Control mice and mice homozygous for Mdr1a/b (Abcb1) or Mdr2 (Abcb4) gene disruption, the latter unable to secrete phospholipids and cholesterol into bile, were treated with griseofulvin to chemically induce protoporphyria. All groups showed dramatically increased PP levels in erythrocytes and liver after griseofulvin treatment. Histologically, massive PP deposits were found in livers of control and Mdr1a/b−/− mice but not in those of Mdr2−/− mice. Serum unesterified cholesterol and phospholipids were increased by griseofulvin because of formation of lipoprotein-X in control and Mdr1a/b−/− mice only. Yet, bile flow was not impaired in griseofulvin-treated mice, and biliary bile salt, phospholipid, and cholesterol secretion rates were significantly increased. Surprisingly, biliary PP excretion was similar in all 3 groups of griseofulvin-treated mice: the observed linear relationship between hepatic and biliary PP concentrations and identical liver-to-bile concentration ratios in treated and untreated mice suggest a passive mode of excretion. In conclusion, the data show that Mdr P-glycoproteins are not critically involved in biliary removal of excess PP and indicate that the presence of biliary lipids is required for formation of intrahepatic PP deposits. (HEPATOLOGY .)

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells.

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fech(m1Pas)/Fech(m1Pas) murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Biliary fibrosis associated with altered bile composition in a mouse model of erythropoietic protoporphyria

Background & Aims: Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP. Methods: Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous ( fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous ( fch/+) and wild-type (+/+) mice. Results: Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid–reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (−12%) and iron (−52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na +-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na +-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice. Conclusions: Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium. GASTROENTEROLOGY 1999;117:696-705

Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy.

Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.