The effects of NAFLD on the liver sinusoidal endothelial cells

By Edward HarrisDept. of Biochemistry

Liver structure overview

Sasse et al. 1992

Hepatocyte

Endothelial cell

Kupffer cell Stellate cell Space of Disse

How to purify SEC, HC, HSC

Liver Perfusion• Anesthetize rat

• Abdominal exposure• Cannulate portal vein• Flush liver• Cut liver from rat and place on funnel• Digest with collagenase in circulation• Separate cells by prep. centrifugation

125I-SA-b-LMWH ENDOCYTOSIS(specific pmol/g cell protein)

0 10 20 30

125I-SA b-UFH ENDOCYTOSIS(specific pmol/g cell protein)

0 50 100 150 200

125I-SA-b-UFH ENDOCYTOSIS(specific CPM/g protein)

0 100 200 300 400 500 600

*

*‡

‡

‡

‡ p < 0.005* p < 0.05

‡

125I-SA-b-UFH ENDOCYTOSIS(specific CPM/g protein)

0 30 60 90 120 150

no Ab

235

174

467

IgG

AN

TI-R

AT

HA

RE

MO

NO

CLO

NA

L A

NTI

BO

DY

AN

TI-H

UM

AN

s19

0-H

AR

E P

OLY

CLO

NA

L A

NIT

BO

DY

No Ab

Pre-immune IgG

Immune IgG

No Ab

Pre-immune IgG

Immune IgG

A Rat SECs

B Rat SECs

no Ab

174

235

467

IgG

C Rat SECs

D 190-hHARE Cells

*

Latest paper to demonstrate purified SECsHARE/STABILIN-2 IS A CLEARANCE RECEPTOR FOR HIGH AND LOW-MOLECULAR WEIGHT HEPARINS

2009 paper

125I-SA-b-UFH (% of total in assay)

0 20 40 60 80 100

2nd try

bloodliverspeenkidneystomachsm. intest.large intest.eyehearttestelung

Injection and clearance of heparin1. Inject radiolabeled Heparin2. Let animal recover for 30 min.3. Harvest organs

Non-alcohol fatty liver disease (NAFLD)Affects up to 85% of the obese

• Defined as >5% of liver weight is from triacylglycerol• Associated with type II diabetes, hypertension, cardiovascular disease• Liver may make some of the TAG• Liver may be a dumping depot for TAG from other tissues• Precursor for steatohepatitis• Increased levels of AST and ALT (aminotransferase enzymes)

NetNebraska.org stats:55% of Nebraskans fit the “obese” categoryLeast fit state in the Union1 in 4 people in the world are overweight and now outnumber hungry people

Qualityhealth.com: 10 fattest cities in America (Nov. 20, 2008)#7 – Scottsbluff, NE 31% obese, 9% T2D

Pathways to Liver illness

Old Age

Damage by high fat diet?

Capillarization of SEC& defenestration

ECM in Space of DisseLiver FibrosisIncreased ALT

pseudocapillarization

Activation of HSC

Cirrhosis

What (cell) starts the cascade of events?

Viral infection

Overall Aims of project1. Measure liver SEC and HC stress as indicated by scavenger receptor function and fenestration in animals on specific fatty acid diets.

2. To determine the degree of ER stress in whole liver and nonparenchymal cells.

3. Determine cell-cell biochemical mediators that promote pro-inflammatory cellular activation

Significance: This “disease” affects ~1/3 of the people in this state

Innovation: No one has really looked at NAFLD from the SEC angle.

Aim 1Measure liver SEC and HC stress as indicated by scavenger

receptor function and fenestration in animals on specific fatty acid diets

60% Lard diet 12% Lard dietPredominant Fat source Lard – 12%

Control dietLard – 60%High fat diet

Protein % 18% 24.2%Carbohydrate% 67% 27.8%Fat % 5.2% 34.7%Fiber % 4.1% 5.5%Total % 100% 100%Energy, calories 3.87 kcal/gm 5.21 kcal/gmFrom protein % 18.6% 18.6%From Carbohydrate % 69.4% 21.4%From Fat % 12% 59.9%Total % 100% 100%

Fenestration

Young & healthy

Old and sickly

1. Visualize by SEM2. Porosity by liposomal transfer

Endocytosis

Purify SEC and HC from both rats

• Hyaluronan for SEC• Orosomucoid for HC

3 Methods that we can use• Injection of labeled ligand • Flush/excision of liver• Perfuse liver, isolate cells

Aim 2ER stress

Mitochondria Peroxisomes

Fatty acid overload

ROS

ER stressATF6CHOPGRP78XBP1 (cut)

Aim 2

Evaluation of ER stress in all 3 cell types: hepatocytes, stellate cells, SECs

qRT-PCR: ATF6, CHOP, GRP78PCR: XBP1

Perfuse livers of rats on both diets

Purify cells

Obtain mRNAs

Use of cDNA arrays to fish for other markers

How liver cells talk to each other

HC

SEC

HSC

Nitric oxide

eNOS

VEGFHGFVEGF

How liver cells may talk to each other during NAFLD

HC

SEC

Activated HSCF-actinTimp1

a smooth muscle actin

Nitric oxide

Fibrosis eNOS

VEGFHGFVEGF

ROS

Mode of action of the reactive oxygen species (ROS)

ROS

X

eNOSIL-6 pathway

Akt/PKB

IKK

NF-kB

Increases in TIMP-1, aSMA, MMP-2, TGF-b and others?

Aim 3: Determine cell-cell biochemical mediators that promote pro-inflammatory cellular activation

4 ways to quantify NO in liver

1. Whole tissue extract. cGMP is a marker for NO bioavailability. 2. eNOS converts L-Arg to NO and L-citrulline. Incubate purified SECs with 3H-Arg and assays for 3H-citrulline.3. Stain cells with 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA; Molecular Probes)4. Co-cultures of activated HSC with SECs and look for response to quiescence.

ROS in hepatocytes in NAFLD vs control

The principle reactive oxygen species we are interested in aresuperoxides (O2-) and peroxides H2O2

These can be measure by exposing cells to 2’7’-dichlorofluorescin diacetate

Hepatocytes from rats on each of the diets will be purified and plated.

Cytokines and other pro-inflammatory mediators

Whole Livers vs Purified Cells

2 primary methods: qRT-PCR (gene expression) and antibody-based kits (protein levels)

Detect known culprits: TIMP-1, MMP-2, TGF-bFish for others that may be cell specific: NF-kB array

SEC, HC purificationSEC, HC culturingEndocytosis assaysRat injectionsClearance strategiesELISAEnzyme assaysRadiolabeling based assaysRNA purificationRT-PCRFluorescence microscopyLiposome productionMALLS

Raising ratsCytokine measurementsNitric oxide chemistryBlood workCo-culturing HC, SECHSC purification, culturingIn vivo imagingqRT-PCRSEM

Recombinant DNA Molecular biologyGene cloningProtein purificationStable cell line productionSugar modificationSPRMutagenesis

Life at OU Proposed project

Questions and Comments?