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?