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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 • • • • • • • 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 A C ANTI-RAT HARE MONOCLONAL ANTIBODY no Ab 174 * ‡ 235 467 * IgG 0 50 100 150 200 125I-SA b-UFH ENDOCYTOSIS (specific pmol/ g cell protein) B Rat SECs no Ab 174 * ‡ 235 ‡ 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 0 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 • • • • • • 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?