Re: PSC - The arteriosclerosis of the bile duct (LONG reply to Nina)

January 29, 2007

Hi Nina;

Although fibrates were not mentioned in the Fickert et al article,

you are correct that they seem to be beneficial in atherosclerosis.

Fibrates are activators of a receptor called the peroxisome-

proliferator activated receptor alpa (PPARa) which controls a host of

genes involved in lipid and cholesterol metabolism, and may help

suppress inflammation and atherosclerosis:

___________________________

Arterioscler Thromb Vasc Biol. 2002 May 1;22(5):717-26.

Pleiotropic actions of peroxisome proliferator-activated receptors in

lipid metabolism and atherosclerosis.

Barbier O, Torra IP, Duguay Y, Blanquart C, Fruchart JC, Glineur C,

Staels B.

UR545 INSERM, Departement d'Atherosclerose, Institut Pasteur de

Lille, and Faculte de Pharmacie, Universite de Lille II, Lille,

France.

Peroxisome proliferator-activated receptors (PPARs) are nuclear

receptors activated by fatty acids and derivatives. Although

PPARalpha mediates the hypolipidemic action of fibrates, PPARgamma is

the receptor for the antidiabetic glitazones. PPARalpha is highly

expressed in tissues such as liver, muscle, kidney, and heart, where

it stimulates the beta-oxidative degradation of fatty acids.

PPARgamma is predominantly expressed in adipose tissues, where it

promotes adipocyte differentiation and lipid storage. PPARbeta/delta

is expressed in a wide range of tissues, and recent findings indicate

a role for this receptor in the control of adipogenesis.

Pharmacological and gene-targeting studies have demonstrated a

physiological role for PPARs in lipid and lipoprotein metabolism.

PPARalpha controls plasma lipid transport by acting on triglyceride

and fatty acid metabolism and by modulating bile acid synthesis and

catabolism in the liver. All 3 PPARs regulate macrophage cholesterol

homeostasis. By enhancing cholesterol efflux, they stimulate the

critical steps of the reverse cholesterol transport pathway. As such,

PPARs control plasma levels of cholesterol and triglycerides, which

constitute major risk factors for coronary heart disease.

Furthermore, PPARalpha and PPARgamma regulate the expression of key

proteins involved in all stages of atherogenesis, such as monocyte

and lymphocyte recruitment to the arterial wall, foam cell formation,

vascular inflammation, and thrombosis. Thus, by regulating gene

transcription, PPARs modulate the onset and evolution of metabolic

disorders predisposing to atherosclerosis and exert direct

antiatherogenic actions at the level of the vascular wall. PMID:

12006382.

___________________________

Fibrates also activate (via PPARa) some bile transporters in the

liver, including Mdr2 (mouse)/MDR3 (human) = ABCB4:

___________________________

J Atheroscler Thromb. 2005;12(4):211-7.

A nuclear receptor-mediated choleretic action of fibrates is

associated with enhanced canalicular membrane fluidity and

transporter activity mediating bile acid-independent bile secretion.

Nishioka T, Hyogo H, Numata Y, Yamaguchi A, Kobuke T, Komichi D,

Nonaka M, Inoue M, Nabeshima Y, Ogi M, Iwamoto K, Ishitobi T, Ajima

T, Chayama K, Tazuma S.

Department of Medicine and Molecular Science, Graduate School of

Biomedical Sciences, Hiroshima University, Hiroshima, Japan.

Fibrates are commonly used lipid-lowering agents that act via

PPARalpha, a member of the nuclear hormone receptor superfamily. The

mechanism(s) of fibrate-induced changes in the hepatic canalicular

membrane and bile lipids are still unknown. Therefore, the aim of

this study was to investigate the influence of fibrates on hepatic

lipid metabolism and to assess the hepatocellular cytoprotective

effect on hepatocyte canalicular membrane. Male ICR mice were fed

standard chow with or without bezafibrate (100 mg/kg) for 6 days. The

expression of canalicular membrane transporters (Mdr2 and Mrp2) was

evaluated by RT-PCR and Western blotting. Canalicular membrane

fluidity was also investigated. Canalicular membrane fluidity was

markedly increased by fibrates. The expression of mdr 2 and mrp2 mRNA

and protein showed a significant increase in fibrate-treated mice.

These results suggested that fibrates improve liver function by

enhancing bile secretion. The mechanism of the choleretic action of

fibrate therapy might involve the enhancement of bile acid-

independent bile secretion, since increased expression of Mdr2 and

Mrp2 was found in fibrate-treated animals. These changes were very

likely mediated by PPARalpha, and the increase of canalicular

membrane fluidity may have been partly associated with enhancement of

this transporter activity. PMID: 16141625.

___________________________

J Lipid Res. 2004 Oct;45(10):1813-25.

Bezafibrate stimulates canalicular localization of NBD-labeled PC in

HepG2 cells by PPARalpha-mediated redistribution of ABCB4.

Shoda J, Inada Y, Tsuji A, Kusama H, Ueda T, Ikegami T, Suzuki H,

Sugiyama Y, Cohen DE, Tanaka N.

Department of Gastroenterology, Institute of Clinical Medicine,

University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305-8575,

Japan. shodaj@...

Fibrates, including bezafibrate (BF), upregulate the expression of

ATP binding cassette protein B4 (ABCB4) through gene transcription in

mice. To determine the effects of BF on the expression levels of

ABCB4 and on the stimulation of biliary phosphatidylcholine (PC)

transport in human HepG2 hepatoblastoma cells, mRNA and protein

levels as well as subcellular localization were investigated in the

cells treated with BF. The canalicular accumulation of a fluorescent

PC was assessed by confocal laser scanning microscopy. Treatment with

300 micromol/l BF for 24 h increased levels of ABCB4 mRNA but not

protein by up to 151%. BF caused redistribution of ABCB4 into

pseudocanaliculi formed between cells. In association with this

redistribution, BF accelerated the accumulation of fluorescent PC in

bile canaliculi (up to 163% of that in nontreated cells). Suppression

of peroxisome proliferator-activated receptor alpha (PPARalpha)

expression by either a small interfering RNA duplex or morpholino

antisense oligonucleotide attenuated the BF-induced redistribution of

ABCB4. These findings suggest that BF may enhance the capacity of

human hepatocytes to direct PC into bile canaliculi via PPARalpha-

mediated redistribution of ABCB4 to the canalicular membrane. This

provides a rationale for the use of BF to improve cholestasis and/or

cholangitis that is attributable to hypofunction of ABCB4.

PMID: 15258199.

___________________________

So fibrates may be directly regulating the phospatidylcholine

transporter that Fickert et al are proposing as a model for primary

sclerosing cholangitis:

" multidrug resistance gene (Mdr2/Abcb4) knockout mice (Mdr2-/-), a

well characterized mouse model that closely mirrors the

macroscopic and microscopic pathology of PSC [8],[11] "

This is a really neat connection between PSC and atherosclerosis.

But, thus far, mutations in the ABCB4 gene have not been found in

PSC, see for example:

___________________________

Hepatology. 2004 Mar;39(3):779-91.

BSEP and MDR3 haplotype structure in healthy Caucasians, primary

biliary cirrhosis and primary sclerosing cholangitis.

i-Magnus C, Kerb R, Fattinger K, Lang T, Anwald B, Kullak-Ublick

GA, Beuers U, Meier PJ

Division of Clinical Pharmacology and Toxicology, University Hospital

Zurich, Ramistrasse 100, A-8091 Zurich, Switzerland.

christiane.pauli@...

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis

(PSC) are characterized by a cholestatic pattern of liver damage,

also observed in hereditary or acquired dysfunction of the

canalicular membrane transporters bile salt export pump (BSEP,

ABCB11) and multidrug resistance protein type 3 (MDR3, ABCB4).

Controversy exists whether a genetically determined dysfunction of

BSEP and MDR3 plays a pathogenic role in PBC and PSC. Therefore, 149

healthy Caucasian control individuals (control group) were compared

to 76 PBC and 46 PSC patients with respect to genetic variations in

BSEP and MDR3. Sequencing spanned approximately 10,000 bp including

promoter and coding regions as well as 50-350 bp of flanking intronic

regions. In all, 46 and 45 variants were identified in BSEP and MDR3,

respectively. No differences between the groups were detected either

in the total number of variants (BSEP: control group: 37, PBC: 37,

PSC: 31; and MDR3: control group: 35; PBC: 32, PSC: 30), or in the

allele frequency of the common variable sites. Furthermore, there

were no significant differences in haplotype distribution and linkage

disequilibrium. In conclusion, this study provides an analysis of

BSEP and MDR3 variant segregation and haplotype structure in a

Caucasian population. Although an impact of rare variants on BSEP and

MDR3 function cannot be ruled out, our data do not support a strong

role of BSEP and MDR3 genetic variations in the pathogenesis of PBC

and PSC. PMID: 14999697.

___________________________

As far as I know, mutations in the human ABCB4 gene have only been

associated with intrahepatic cholestasis of pregnancy:

___________________________

Hepatology. 2007 Jan;45(1):150-8.

Linkage between a new splicing site mutation in the MDR3 alias ABCB4

gene and intrahepatic cholestasis of pregnancy.

Schneider G, Paus TC, Kullak-Ublick GA, Meier PJ, Wienker TF, Lang T,

van de Vondel P, Sauerbruch T, Reichel C.

Department of Internal Medicine I, University of Bonn, Bonn, Germany.

Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus

and elevated bile acid serum concentrations in late pregnancy.

Splicing mutations have been described in the multidrug resistance p-

glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women.

Pedigrees studied were not large enough for linkage analysis. Ninety-

seven family members of a woman with proven ICP were asked about

pruritus in earlier pregnancies, birth complications and symptomatic

gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1)

gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were

analyzed in 55 relatives. We identified a dominant mode of

inheritance with female restricted expression and a new intronic MDR3

mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe

deletion via cryptic splicing site activation. Linkage analysis of

the ICP trait versus this intragenic MDR3 variant yielded a LOD score

of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an

unknown locus gave a posterior probability of >0.9966 in favor of

MDR3 as causative ICP locus. During the episode of ICP the median

gamma-glutamyl transpeptidase (gamma-GT) activity was 10 U/l (95% CI,

6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported

in seven heterozygous women (22 pregnancies) and none in five women

(14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease

was more prevalent in heterozygous relatives (7/21) than in relatives

without the mutation (1/34), (P = 0.00341). CONCLUSION: This study

demonstrates that splicing mutations in the MDR3 gene can cause ICP

with normal gamma-GT and may be associated with stillbirths and

gallstone disease. PMID: 17187437.

___________________________

and one type of progressive familial intrahepatic cholestasis:

___________________________

Pflugers Arch. 2006 Apr 19; [Epub ahead of print]

Function and pathophysiological importance of ABCB4 (MDR3 P-

glycoprotein).

Oude Elferink RP, usma CC

AMC Liver Center, Academic Medical Center, Meibergdreef 69-71,

1105AZ, Amsterdam, The Netherlands, r.p.oude-elferink@....

Like several other ATP-binding cassette (ABC) transporters, ABCB4 is

a lipid translocator. It translocates phosphatidylcholine (PC) from

the inner to the outer leaflet of the canalicular membrane of the

hepatocyte. Its function is quite crucial as evidenced by a severe

liver disease, progressive familial intrahepatic cholestasis type 3,

which develops in persons with ABCB4 deficiency. Translocation of PC

makes the phospholipid available for extraction into the canalicular

lumen by bile salts. The primary function of biliary phospholipid

excretion is to protect the membranes of cells facing the biliary

tree against these bile salts: the uptake of PC in bile salt micelles

reduces the detergent activity of these micelles. In this review, we

will discuss the functional aspects of ABCB4 and the regulation of

its expression. Furthermore, we will describe the clinical and

biochemical consequences of complete and partial deficiency of ABCB4

function. PMID: 16622704.

__________________

Apparently, the clinical consequences can vary depending on the

severity of the mutation, and how much of the function of the

phospholipid transporter is impaired. Anyhow, this is why Fickert et

al suggest focusing attention on " unusual " cases of PSC (e.g., small

duct PSC, young women suffering from PSC, PSC patients with

gallstones).

I hope this answers some of your questions. Basically, we don't know

what the key PSC susceptibility gene is right now ... some suggest it

may simply be a specific variant of the cystic fibrosis transmembrane

conductance regulator gene:

_____________________________

Hum Genet. 2003 Aug;113(3):286-92.

Increased prevalence of CFTR mutations and variants and decreased

chloride secretion in primary sclerosing cholangitis.

Sheth S, Shea JC, Bishop MD, Chopra S, Regan MM, Malmberg E,

C, Ricci R, Tsui LC, Durie PR, Zielenski J, Freedman SD

Department of Medicine, Beth Israel Deaconess Medical Center/Harvard

Medical School, Dana 532, 330 Brookline Avenue, Boston, MA 02215, USA.

Primary sclerosing cholangitis (PSC) and cystic fibrosis (CF) are

both slowly progressive cholestatic liver diseases characterized by

fibro-obliterative inflammation of the biliary tract. We hypothesized

that dysfunction of the CF gene product, cystic fibrosis

transmembrane conductance regulator (CFTR), may explain why a subset

of patients with inflammatory bowel disease develop PSC. We

prospectively evaluated CFTR genotype and phenotype in patients with

PSC ( n=19) compared with patients with inflammatory bowel disease

and no liver disease ( n=18), primary biliary cirrhosis ( n=17), CF (

n=81), and healthy controls ( n=51). Genetic analysis of the CFTR

gene in PSC patients compared with disease controls (primary biliary

cirrhosis and inflammatory bowel disease) demonstrated a

significantly increased number of mutations/variants in the PSC group

(37% vs 8.6% of disease controls, P=0.02). None of the PSC patients

carried two mutations/variants. Of PSC patients, 89% carried the

1540G-variant-containing genotypes (resulting in decreased functional

CFTR) compared with 57% of disease controls ( P=0.03). Only one of 19

PSC patients had neither a CFTR mutation nor the 1540G variant. CFTR

chloride channel function assessed by nasal potential difference

testing demonstrated a reduced median isoproterenol response of 14 mV

in PSC patients compared with 19 mV in disease controls ( P=0.04) and

21 mV in healthy controls ( P=0.003). These data indicate that there

is an increased prevalence of CFTR abnormalities in PSC as

demonstrated by molecular and functional analyses and that these

abnormalities may contribute to the development of PSC in a subset of

patients with inflammatory bowel disease. PMID: 12783301.

_____________________________

In this background, a trigger might be the occurrence of IBD, much as

when cftr (-/-) mice only develeop sclerosing cholangitis when given

colitis:

______________________________

Am J Physiol Gastrointest Liver Physiol. 2004 Aug;287(2):G491-6.

Induction of colitis in cftr-/- mice results in bile duct injury.

Blanco PG, Zaman MM, Junaidi O, Sheth S, Yantiss RK, Nasser IA,

Freedman SD

Beth Israel Deaconess Medical Center, 330 Brookline Ave., Dana 501,

Boston, MA 02215, USA.

It is unknown why some patients with inflammatory bowel disease

develop primary sclerosing cholangitis. We have recently shown that

patients with primary sclerosing cholangitis have an increased

prevalence of mutations in the gene responsible for cystic fibrosis

(CFTR) compared with individuals with inflammatory bowel disease

alone. Our aim was to examine whether induction of colitis by oral

dextran leads to bile duct injury in mice heterozygous or homozygous

for mutations in CFTR. The effect of oral administration of

docosahexaenoic acid to correct a fatty acid imbalance associated

with cystic fibrosis was also examined to determine whether this

would prevent bile duct inflammation. Wild-type mice and mice

heterozygous and homozygous for CFTR mutations were given dextran

orally for 14 days to induce colitis. Bile duct injury was

quantitated by blinded histological scoring and measurement of serum

alkaline phosphatase activity. The effect of pretreatment with

docosahexaenoic acid for 7 days was examined. Treatment of mice with

100 mg dextran/day for 9 days followed by 85 mg/day for 5 days

resulted in a significant increase in bile duct injury as determined

by histological scoring in homozygous cystic fibrosis mice compared

with wild-type mice (P = 0.005). The bile duct injury seen in cystic

fibrosis mice was reflected in a threefold increase in serum alkaline

phosphatase (P = 0.0006). Pretreatment with oral docosahexaenoic acid

decreased both histological evidence of bile duct injury and serum

alkaline phosphatase levels. In the setting of colitis, loss of CFTR

function leads to bile duct injury. PMID: 15064232.

__________________________

Again, this seems to be tied-in with PPARa:

__________________________

J Pediatr Gastroenterol Nutr. 2006 Mar;42(3):275-81.

Decreased peroxisome proliferator activated receptor alpha is

associated with bile duct injury in cystic fibrosis transmembrane

conductance regulator-/- mice.

Pall H, Zaman MM, Andersson C, Freedman SD

Division of Pediatric Gastroenterology, Children's Hospital Boston,

Harvard Medical School, Boston, Massachusetts, USA.

BACKGROUND: Primary sclerosing cholangitis (PSC) is associated with

mutations in the cystic fibrosis transmembrane conductance regulator

(CFTR) gene. As proof of concept that CFTR dysfunction plays a role

in PSC, induction of colitis in cftr mice results in bile duct injury

that can be prevented by pretreatment with docosahexaenoic acid

(DHA). OBJECTIVES: Determine whether 1) CFTR dysfunction in cftr mice

through a reduction in peroxisome proliferator activated receptor

(PPAR)alpha or gamma leads to bile duct injury and 2) whether DHA

prevents bile duct injury through an increase in PPAR. METHODS: Cftr

and wild-type (WT) mice were treated with dextran sodium sulfate

(DSS) to induce colitis with or without pretreatment with oral DHA.

PPARalpha and gamma as well as tumor necrosis factor (TNF)alpha were

analyzed in liver tissue. PPARalpha mice were also treated with DSS

and histology examined. RESULTS: PPARgamma mRNA levels were low, with

DSS suppressing mRNA levels equally in WT and cftr mice. PPARalpha

levels were no different between cftr and WT litter mates by reverse-

transcription polymerase chain reaction. After DSS, WT mice showed a

9.3-fold increase in PPARalpha mRNA levels and increased nuclear

localization compared with no DSS (P < 0.05), with no increase seen

in cftr mice. This was not caused by changes in TNFalpha. DHA

treatment led to 7.0-fold increase in PPARalpha mRNA levels in cftr

mice (P < 0.01). PPARalpha mice treated with DSS did not develop bile

duct injury, indicating that PPARalpha alone is not sufficient to

cause bile duct inflammation. CONCLUSION: DSS induced bile duct

injury in cftr mice is associated with a defect in PPARalpha

expression, which is reversed by DHA. PMID: 16540796.

_____________________________

Based on this and other reading, we've encouraged our son to take

fish oils, which may in part be serving to activate PPARa. I think

that fish oils have a better safety profile than fibrates.

Best regards,

Dave

(father of (21); PSC 07/03; UC 08/03)

January 30, 2007

, thank you so much for your terrific reply. I've read through it

briefly and while it will of course take several more readings to fully

digest I hope you know how much I and I'm sure we all really appreciate

the strong scientific background you bring to these discussions.

Ultimately Sam and I keep going back and forth on fibrates for just

that reason - safety - but the fish oil he tried initially didn't seem

to do anything and the newer stuff, which is 450/90 DHA/EPA, doesn't

seem to be making much of a difference in the Alk Phos numbers either.

Anyway, this is really helpful for our decision. Thanks so much.

Nina

> Based on this and other reading, we've encouraged our son to take

> fish oils, which may in part be serving to activate PPARa. I think

> that fish oils have a better safety profile than fibrates.

>

> Best regards,

>

> Dave

> (father of (21); PSC 07/03; UC 08/03)

>

January 30, 2007