?ACKNOWLEDGMENT?TABLE OF CONTENTSAcknowledgment 2Table of Contents 3List of Abbreviations 5List of Tables 6List of Figures 7Abstract 8Résumé 9Chapter I: Introduction 101. Hepatic fibrosis 102. Architecture of the normal liver 112.1. Anatomy 112.

2. Function 122.3.

Cells within the Liver 122.4. The liver in health and disease 133. Pathway of liver fibrosis 143.1. Composition and remodeling of ECM 143.

2. Immune response 143.3.

Profibrotic mediators 144. Regression of fibrosis 145. Effect of Statins on hepatic fibrosis 145.1. Definition 145.2.

Pathway 145.3. Role in inflammation and fibrosis 14Aim of the Project 14Chapter II: Materials and methods 151. Animals 152. Experimental Design 152.1 Carbon tetrachloride- (CCL4-) induced liver injury 153. SR 154.

Immunohistochemistry staining of hepatic ?SMA 155. ALT and AST detection 156. RNA Extraction 157. Reverse transcription-PCR 158. Real-Time PCR 169. Statistical analysis 16Chapter III: Results 17Chapter IV: Discussion and Conclusion 18Chapter V: Future perspectives 19References 20?LIST OF ABBREVIATIONS?LIST OF TABLES?LIST OF FIGURESFigure 1: Structure of the healthy liver.

11Figure 2: Cellular modifications in the sinusoid during liver injury 9. 13?ABSTRACT?RÉSUMÉ?CHAPTER I: INTRODUCTIONLiver disease is a major cause of morbidity and mortality worldwide, and the sequent loss of liver function is a critical clinical challenge. There are many different types of liver disease, which can be broadly grouped into three categories: chronic liver disease caused by metabolic dysfunction, acute liver failure that does not damage normal tissue structure, however is related to direct injury and rapid deterioration of hepatic function. Also, chronic liver failure that is associated with widespread tissue damage and scar-based remodeling, which can eventually lead to end-stage cirrhosis and hepatocellular carcinoma 1.

Hepatic damage can be induced by several factors including viral infection (hepatitis B and C), alcohol abuse, autoimmune hepatitis and chronic cholangiopathies. Also accelerated liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is associated with obesity rates. This situation can cause chronic hepatic inflammation and deregulated wound healing process in the liver, which, if prolonged, can lead to fibrosis 2.1. Hepatic fibrosisHepatic fibrosis is the main complication of chronic liver failure and characterized by the excessive accumulation of an altered extracellular matrix, that is extremely rich in type I and III collagens. Deposition of scar tissue results from a wound healing response that occurs to maintain liver integrity after several insults from various biochemical metabolites 3. However, the continuous unbalanced synthesis of matrix protein and degradation leads to an incomplete matrix remodeling and irreversible cirrhosis 4.Cirrhosis is a late stage condition in which the architecture of the liver becomes abnormal, the function of hepatocytes is reduced, and the hepatic blood ?ow is altered due to vascularized ?brotic septa surrounding regenerating nodules.

Liver cirrhosis results in multiple complications such as coagulation defect and portal hypertension, including ascites, variceal bleeding, renal failure, hepatic encephalopathy, bacterial peritonitis and finally hepatocellular carcinoma 3.2. Architecture of the normal liver2.1. Anatomy The liver is the heaviest visceral organ in the body, expressing 2–5% of body weight and exhibits an iterative, multicellular architecture.

The organ is divided into four lobes; yet, the liver lobule represents its functional units.Each lobule is composed of hexagonal cords of hepatocytes arranged around a central vein that drain into the large hepatic vein. The corners of the hexagon constitute the portal triad consisting of a portal vein, hepatic artery and biliary duct (Figure 1-A).

Within a lobule, two afferent vessels supply hepatic blood: the hepatic artery and the portal vein, and flows in specialized sinusoidal vessels towards the central vein 1.The hepatic sinusoid is a complex vascular channel built from specialized fenestrated endothelial cells of the liver also it is the residence of the hepatic macrophages named Kupffer cells. Stellate cells are located in the sub-endothelial space known as the space of Disse that separates the hepatocyte cords from the blood and the sinusoids (Figure 1-B). Bile, that is produced and excreted by hepatocytes into the bile canaliculi, flows in the opposite direction to sinusoidal blood flow towards the intrahepatic bile duct, which is lined by epithelial cells called cholangiocytes 5. Figure 1: Structure of the healthy liver 5.

(A) Geometric organization of the hepatic lobule, the functional unit of the liver. (B) A schematic representation of a sinusoid within the liver and the corresponding location of different hepatic cells. 2.2. FunctionThe liver exhibits many functions in the body, including filtration of the blood, endocrine control of growth signaling pathways and biliary excretion (bile salts and bicarbonate) that facilitates digestion of fats and lipids 1.

The liver also provides immune system support, detoxifies chemicals such as xenobiotics, and metabolizes drugs and macronutrient supplying the body with the needed energy.Carbohydrate storage as glycogen and glucose manufacture via the gluconeogenic pathway is the most critical liver function, in addition to cholesterol homeostasis, lipids oxidation, and storage of excess lipid in other tissues, such as adipose. Finally, the liver is a major producer of the proteins secreted in the blood, their conversion into amino acids, and removal of nitrogen in the form of urea metabolism 6. 2.3.

Cells within the liverThere are four major cell types that play different roles in order to allow the proper functioning of the liver.2.3.1. Hepatocytes2.3.

2. Kuppfer cellsKupffer cells are non-parenchymal, resident macrophages which are different from in?ltrating macrophages. They are positioned through the sinusoidal endothelial cells and represent 15% of the total hepatic cells. Kupffer cells are important phagocytes in the liver; they help the innate immune response by scavenging microorganisms that reach the sinusoidal vessels, regulating of inflammatory processes, and finally by removing immune complexes, blood debris and toxic substances. Moreover, kupffer cells regulate iron, bilirubin and cholesterol metabolism.Furthermore, to be activated, these cells express several receptors; for instance receptor-mediated endocytosis, Fc receptor and Toll-like receptor 4 (TLR4).

They also express CD14 and CD68 as surface markers, yet they are negative for CX3CR1. Activation by LPS, DAMPs or complement component leads kypffer cells to release cytokines and chemokines such as CCL2, CCL5, TNF-?, IL-1, IL-6, and reactive oxygen species, promoting the recruitment and activation of other pro-inflammatory cells. In addition, kupffer cells stimulate anti-inflammatory cells by secreting IL-10 specially at the acute phase of liver damage 7, 8. 2.

3.3. Sinusoidal endothelial cells (SEC)Liver sinusoidal endothelial cells (LSECs) form the wall of liver sinusoid that separate hepatocytes from the blood. These cells have the highest percentage of the non-parenshymal hepatic cells; comprising about 15% of liver cells and 3% of hepatic volume. Upon their differentiation into adult LSECs, they gain markers such as CD4, CD32 and ICAM-1. Yet, some of these markers are similar to other cells including endothelial and hematopoietic cells but none of them is specific for LSECs 9.

LSECs represent a permeable barrier which displays distinctive structural features that make them different from other endothelial cells. In fact, not having a basal membrane neither a diaphragm yet possessing of fenestrae make these cells the most permeable cells with the highest endocytosis capacity of any cell in the body 10. Also, LSECs have critical physiological, scavenger and immunological functions 2.3.4.

Hepatic Stellate Cells (HSCs)2.4. The liver in health and diseaseGenerally, disturbance of liver’s morphology and function initiate with the injured hepatocytes, once they stimulate the pro-inflammatory pathway. Activated kupffer cells release pro-fibrotic mediators that change the phenotype of HSCs from quiescent to activated cells, resulting in scar formation. The accumulation of extracellular matrix proteins is responsible for the disappearance of endothelial fenestrae and the loss of hepatocytes microvilli (Figure 2) 11. Figure 2: Cellular modifications in the sinusoid during liver injury 11.3. Pathway of liver fibrosis3.

1. Composition and remodeling of ECM3.2. Immune response3.2.1 Activation of HSC3.2.

2 Hepatocytes apoptosis3.3. Profibrotic mediators4. Regression of fibrosis5. Effect of Statins on hepatic fibrosis5.1. Definition5.

2. Pathway 5.3.

Role in inflammation and fibrosisAIM OF THE PROJECT?CHAPTER II: MATERIALS AND METHODS1. Animals Eleven-week-old male C57BL/6J mice weighing 20-30 g were purchased from *** Laboratories and housed in a pathogen-free environment. All experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the American University of Beirut (AUB) following the ‘Guide for the care and use of laboratory animals’ and the “US Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research and Training”.

2. Experimental Design2.1 Carbon tetrachloride- (CCL4-) induced liver injuryEach mouse was given two intraperitoneal injections of 0.6ml/kg CCl4 (**) mixed with mineral oil (**) at the ratio of 1:10, for – consecutive weeks. The control group was injected with mineral oil.3. SR4. Immunohistochemistry staining of hepatic ?SMA5.

ALT and AST detectionBlood samples were collected on the day of sacrifice. The samples were centrifuged at 15000 xg for 15 minutes at 4 ºC. Serum ALT and AST levels were detected using *** kit (***) by ***6. RNA Extraction7. Reverse transcription-PCRReverse transcription was performed on 1µg of total RNA in a final 20 µl volume using the *** kit () this included creating a negative RT control without reverse transcriptase. The cycle begins at 25°C for 10 min, 37°C for 2 hours, 85°C for 5 min, and ends at 4°C, using the RT-PCR machine (Bio-Rad Laboratories, California, USA).

The cDNA samples were stored at -20°C.8. Real-Time PCR9. Statistical analysisAnimals were randomly selected for the control and treatment group.

All results were expressed as the means ± SEM. Differences between groups were analyzed by the Mann-Whitney test, using “GraphPad Prism” software. The p values for p