Branko Stefanovic, Ph.D. Florida State University College of Medicine Dept. of Biomedical Sciences 1115 West Call Street Tallahassee, FL 32306-4300 Office: (850) 644-7600 Dr. Stefanovic's Faculty Profile
Research Interests
Liver fibrosis (with its end stage, cirrhosis) is the 8th leading cause of death in the USA. There is no cure for liver fibrosis. Main causes of liver fibrosis are alcohol abuse, infection with hepatitis B or C viruses, NASH syndrome, cholestasis of various etiologies and genetic diseases. 95% of liver cancers appear in cirrhotic livers. Other major complications of liver fibrosis include jaundice, varicose bleeding, portal hypertension and ascites. Liver fibrosis is characterized by deposition of collagen type I within the space of Disse of the liver sinusoids. In normal liver type I collagen represents 1% of total liver proteins, while in cirrhotic liver it can account for 50% of the total proteins. Type I collagen is synthesized in the liver by a minor cell population called hepatic stellate cells. Major liver cells, hepatocytes, which are responsible for almost all liver functions, have a great potential to regenerate. However, in fibrosis liver regeneration is deficient because type I collagen is not a natural matrix within which hepatocytes can grow. Patients with liver fibrosis die because of uncontroled synthesis of type I collagen by the activated hepatic stellate cells (HSCs). Therefore, to understand and help find a cure for liver fibrosis my lab studies activation of hepatic stellate cells, regulation of expression of genes encoding for type I.collagen and conection between inflammation and fibrosis.
Current Projects
Discovery of novel genes involved in activation of HSCs Hepatic stellate cells (HSCs, also named Ito cells, lipocytes, or fat-storing cells) are the major cell type responsible for collagen synthesis in the cirrhotic liver. In normal liver, quiescent HSCs store vitamin, but only express trace amounts of type I collagen. Upon a fibrogenic stimulus, HSCs become activated, a process in which they lose retinoid droplets, proliferate, change morphologically into myofibroblasts, and increase their synthesis of extracellular matrix proteins. We routinely isolate HSCs from rat livers to study their activation in vitro. We also use animal models of liver injury to study HSCs activation in vivo. Characterization of genes responsible for activation of HSCs is another major project in my lab. To discover genes relevant for activation of HSCs my lab has obtained three DNA microarray data sets on gene expression profiles in HSCs and cirrhotic livers. The first DNA microarray compared gene expression in normal and cirrhotic human livers. This expression profile allowed us to identify upregulated and down regulated genes in alcohol or hepatitis induced cirrhosis. The second DNA microarray compared gene expression between isolated quiescent HSCs and activated HSCs. These results allowed us to identify genes which are expressed during activation of HSCs. The third data set is a comparison of gene expression between proliferating and senescent HSCs. This allowed us to identify genes responsible for proliferation of HSCs. Based on these results we identified hundreds of novel and relevant genes, which include transcription factors, RNA binding proteins, cytokines, chemokines and regulators of signal transduction. The function of these genes will be analyzed in HCSs and in animal models of liver fibrosis using techniques for analysis of mRNA and proteins. In addition, knock-out mice will be obtained for some of these genes to study their role in the whole organism. Regulation of expression of type I collagen Biosynthesis of type I collagen is a complex process, which requires the coordinated expression of two genes, collagen  α1(I) and collagen α2(I). Polypeptides encoded by these genes fold into heterotrimeric protein, type I collagen. The main step in regulation of expression of these genes is binding of RNA-binding proteins to collagen mRNAs. These RNA binding proteins recognize a unique structure called the 5’ stem-loop. Binding of the protein factors increases in activation of HSCs, which leads to an increased synthesis of type I collagen. We have discovered this mechanism several years ago and described its functional significance in several papers. Cloning and characterization of the regulatory proteins which bind collagen 5’ stem-loop is one major project in my lab. To this goal we use RNA-affinity purification techniques, expression screening of cDNA libraries and methods for studing RNA-protein interactions. Recently, we have developed a knock-in mouse in which we have mutated the 5’ stem-loop in the context of the endogenous collagen α1(I) gene. This will enable us to study the unique aspect of collagen gene regulation in vivo and in animal models of liver fibrosis. When cloned, the 5’ stemloop binding proteins will be characterized as a major step towards developing specific drugs for liver fibrosis, as well as fibrosis of other organs. Inflammation and liver fibrosis The causative agents of liver fibrosis induce chronic inflammation and prolonged inflammation usually precedes fibrosis. The molecular events linking inflammation and fibrosis are poorly understood. KC is a proinflamatory chemokine, expression of which is upregulated in patients with alcoholic cirrhosis and acute alcoholic hepatitis. The only well studied function of KC is chemotaxis of neutrophils to the site of tissue injury. We have shown that activated HSCs express KC, as well as its receptor CXCR2, suggesting that they can be stimulated in an autocrine manner. In the whole liver, expression of KC gene is regulated by KC protein in a positive feedback manner. If triggered, this positive feedback results in accelerated liver injury with increased expression of proinflammatory and profibrotic genes. Thus, expression of KC must be tightly controlled and its upregulation may be of relevance for development of liver failure in alcoholic subjects. This project is aimed to further elucidate the role of KC in liver fibrosis. We constructed a recombinant adenovirus containing the KC gene, which will allow us to specifically express KC in the liver. After injection into mice the effects of KC on the liver will be examined by histology, by measurment of liver enzymes and by expression of profibrotic and proinflamatory genes. Comprehensive changes in gene expression induced by overexpression of KC will be analyzed by DNA microarray technology. A preliminary DNA microarray experiment indicated that overexpression of KC resulted in upregulation of 1000 genes and downregulation of 800 genes in the mouse liver. The expression of the highest upregulated gene was increased 300 fold, while the expression of the most downregulated gene was decreased 800 fold, suggesting dramatic changes. By this approach novel genes involved in linking inflammation and fibrosis will be discovered. They will be subjected to further study as possible targets for drug development.
Current Laboratory Members
Dr. Feng Jiang, postdoc Lela Stefanovic, lab. tech Dillon Fritz, grad. student Mary Cae, grad student
Selected References
Stefanovic B. New insights into regulation of type I collagen expression. J. Biol. Sci. 2004. In press. Lela Stefanovic, Chad E. Stephens, David Boykin, Branko Stefanovic.  Inhibitory effect of dicationic diphenylfurans on production of type I collagen by human fibroblasts and activated hepatic stellate cells. 2004. J. Biol. Sci. In press. Stefanovic B, Rippe RA. Collagen Gene Regulation in the Hepatic Stellate Cell. In: Molecular Mechanisms of Liver Disease. Oxford Press & IBH (India) & M/s Sc Pub (USA). 2004. Stefanovic B, Stefanovic L, Schnabl B, Bataller R, Brenner DA. TRAM2 Protein Interacts with Endoplasmic Reticulum Ca2_ Pump Serca2b and Is Necessary for Collagen Type I Synthesis. Mol.Cell.Biol., 2004 Feb, p. 1758-1768, Vol. 24, No. 4. Rippe RA, Stefanovic B. Techniques to Assess the Molecular Mechanisms Controlling Gene Regulation. In: Fibrosis Research: Methods and protocols. Varga J, Brenner DA, Phan SH (Editors). 2004. The Humana Press Inc, Totowa, NJ.