Transcript The effect of NAFLD on the sinusoidal endothelial cells

The effects of NAFLD on the liver
sinusoidal endothelial cells
By Edward Harris
Dept. of Biochemistry
Liver structure overview
Kupffer cell
Stellate cell
Space of Disse
Hepatocyte
Endothelial cell
Sasse et al. 1992
How to purify SEC, HC, HSC
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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
Liver
Perfusion
Latest paper to demonstrate purified
SECs
2009 paper HARE/STABILIN-2 IS A CLEARANCE RECEPTOR FOR
HIGH AND LOW-MOLECULAR WEIGHT HEPARINS
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C
ANTI-RAT HARE MONOCLONAL ANTIBODY
no Ab
174
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235
467
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IgG
0
50
100
150
200
125I-SA b-UFH ENDOCYTOSIS
(specific pmol/ g cell protein)
B
Rat SECs
no Ab
174
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235
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467
IgG
0
10
20
30
125I-SA-b-LMWH ENDOCYTOSIS
(specific pmol/ g cell protein)
0.05
*‡ pp<<0.005
ANTI-HUMAN s190-HARE POLYCLONAL ANITBODY
Rat SECs
Rat SECs
No Ab
Pre-immune IgG
Immune IgG
0
30
125
60
90
120
150
I-SA-b-UFH ENDOCYTOSIS
(specific CPM/ g protein)
D
190-hHARE Cells
No Ab
Pre-immune IgG
‡
0
Immune IgG
100 200 300 400 500 600
125
I-SA-b-UFH ENDOCYTOSIS
(specific CPM/ g protein)
Injection and clearance of heparin
1. Inject radiolabeled Heparin
2. Let animal recover for 30 min.
3. Harvest organs
blood
liver
speen
kidney
stomach
sm. intest.
large intest.
eye
heart
teste
lung
2nd try
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20
40
125
60
80
I-SA-b-UFH
(% of total in assay)
100
Non-alcohol fatty liver disease (NAFLD)
Affects up to 85% of the obese
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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” category
Least fit state in the Union
1 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
Viral infection
pseudocapillarization
Damage by
high fat diet?
Capillarization of SEC
& defenestration
ECM in Space of Disse
Activation
of HSC
Liver Fibrosis
Increased ALT
Cirrhosis
What (cell) starts the cascade of events?
Overall Aims of project
1. 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 1
Measure liver SEC and HC stress as indicated by scavenger
receptor function and fenestration in animals on specific fatty acid diets
60% Lard diet
Predominant Fat source
Protein %
Carbohydrate%
Fat %
Fiber %
Total %
Energy, calories
From protein %
From Carbohydrate %
From Fat %
Total %
Lard – 12%
Control diet
18%
67%
5.2%
4.1%
100%
3.87 kcal/gm
18.6%
69.4%
12%
100%
Lard – 60%
High fat diet
24.2%
27.8%
34.7%
5.5%
100%
5.21 kcal/gm
18.6%
21.4%
59.9%
100%
12% Lard diet
Fenestration
Endocytosis
Purify SEC and HC from both rats
Young & healthy
Old and sickly
1. Visualize by SEM
2. Porosity by liposomal transfer
• 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 2
ER stress
Fatty acid overload
Mitochondria
Peroxisomes
ROS
ER stress
ATF6
CHOP
GRP78
XBP1 (cut)
Aim 2
Evaluation of ER stress in all 3 cell types: hepatocytes, stellate cells, SECs
Perfuse livers of rats on both diets
Purify cells
Obtain mRNAs
qRT-PCR: ATF6, CHOP, GRP78
PCR: XBP1
Use of cDNA arrays to fish for other markers
How liver cells talk to each other
HC
HSC
Nitric oxide
VEGF
VEGF
eNOS
SEC
HGF
How liver cells may talk to each other
during NAFLD
Activated HSC
F-actin
Timp1
a smooth muscle
actin
HC
Nitric oxide
ROS
VEGF
VEGF
Fibrosis
eNOS
SEC
HGF
Mode of action of the reactive oxygen species
(ROS)
ROS
X
IL-6 pathway
eNOS
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 are
superoxides (O2-) and peroxides H2O2
Hepatocytes from rats on each of the diets will be purified and plated.
These can be measure by exposing cells to 2’7’-dichlorofluorescin diacetate
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-b
Fish for others that may be cell specific: NF-kB array
Life at OU
Recombinant DNA Molecular biology
Gene cloning
Protein purification
Stable cell line production
Sugar modification
SPR
Mutagenesis
Proposed project
SEC, HC purification
SEC, HC culturing
Endocytosis assays
Rat injections
Clearance strategies
ELISA
Enzyme assays
Radiolabeling based assays
RNA purification
RT-PCR
Fluorescence microscopy
Liposome production
MALLS
Raising rats
Cytokine measurements
Nitric oxide chemistry
Blood work
Co-culturing HC, SEC
HSC purification, culturing
In vivo imaging
qRT-PCR
SEM
Questions and Comments?