Research Interests

Molecular Mechanisms involved in liver fibrosis: Alcohol metabolism by the liver results in the formation of acetaldehyde, free radicals and several other aldehydes (malonyldialdehyde [MDA] and 5 hydroxynonenal [5HN] that are derived from lipid peroxidation. All of these contribute to excess collagen deposition in the liver and the development of liver fibrosis, an important component of cirrhosis. Hepatic stellate cells (HSC) are the main cells involved in collagen deposition in the liver. We have shown that acetaldehyde up-regulates the expression of both type I collagen genes in HSC by a hydrogen peroxide-dependent mechanism. We have localized an acetaldehyde-responsive element within the promoter of the a2(I) collagen gene and demonstrated that there are two distinct types of protein DNA complexes. An inhibitor complex whose degradation is enhanced by acetaldehyde and an activator complex that transactivates the expression of the gene. DNA microarray studies revealed that acetaldehyde primes the cells to respond to transforming growth factor-b1 (TGF-b1). We are currently investigating molecular mechanisms whereby acetaldehyde induces the formation of H2O2, TGF-b1 and a discrete family of gene products involved in binding of latent TGF-b1 to the plasma membrane of HSC, and convert the inactive cytokine to active TGF-b1. Based on our findings we are attempting to develop novel therapies to prevent and/or revert liver fibrosis.

Role of matrix metalloproteinases (MMPs) in liver fibrosis: MMPs are involved in collagen remodeling and play a key role in the development of liver fibrosis. Liver fibrosis results from an imbalance between the amount of collagen produced and that degraded by MMPs. Our studies pertaining to the expression of MMP-13, a metalloproteinase that degrades native type I collagen, revealed that this enzyme functions as a scavenger enzyme. Thus, MMP-13 is involved in degradation of collagen after injury and precedes the accumulation of newly synthesized collagen by fibroblasts (in a model of skin wound healing) and HSC. We further showed that collagen and MMP-13 are reciprocally modulated. Thus, when cells produce collagen they express little MMP-13 and vice versa. We are currently investigating signal transduction pathways involved in MMP-13 regulation by TGF-b1 and platelet-derived growth factor.

Long-term culture of functional hepatocytes: We developed a co-culture system containing rat hepatocytes and an HSC clone isolated in our laboratory. In this co-culture system hepatocytes have been maintained alive and functional for 6-7 months. These co-cultures express albumin, fibrinogen, a1-antitrypsin and tyrosine amino transferase, genes expressed by adult hepatocytes. However, the cells also express a-fetoprotein a protein expressed by fetal hepatocytes and hepatomas. We are currently exploring whether a similar co-culture with human hepatocytes can be developed using the same culture conditions used for rat cells. If successful, the culture system could be used to determine whether the hepatitis C virus can replicate and explore new forms of anti-viral therapy. Thus far, there are no reliable methods to replicate HCV in culture. In addition, maintaining the function of adult human hepatocytes in culture could allow us to generate sufficient cells to develop an artificial liver for the treatment of acute liver failure or to sustain the liver function of patients awaiting a liver transplant.

Laminin-binding proteins of hepatocytes and hepatomas: Hepatomas are known to express a laminin-binding protein of 68 kDa. In several tumors, the expression of this laminin binding protein has been associated with metastatic potential. We have isolated a high affinity laminin-binding protein from the cell surface of rat hepatocytes that differs in molecular weight and laminin affinity from that found in tumors. When removed from the cell surface by procedures that do not damage cell function, hepatocytes lose their capacity to bind to laminin. However, laminin binding can be reconstituted after incubation of the hepatocytes with the isolated laminin-binding protein in the presence of divalent cations. Although the same laminin binding protein is expressed by hepatomas, nonetheless it is not localized to the plasma membrane and cannot reconstitute laminin-binding of hepatomas stripped of their 68 kDa laminin-binding protein. Similarly, laminin binding of normal hepatocytes depleted of their laminin-binding protein cannot be reconstituted with the 68 kDa laminin binding protein of hepatomas. We have recently demonstrated that the prior injection of the laminin binding protein of normal hepatocytes into the portal vein prevents the implantation of hepatomas in the liver. We are currently attempting to purify the protein, clone the gene and express the recombinant protein. We would like to determine whether this normal protein is able to prevent metastatic growth of tumors implanted subcutaneously in Scid mice.

Selected Publications (Three most recent):

1. Yanase M, Ikeda H, Matsui A, Noiri E, Tomiya T, Arai M, Inoue Y, Tejima K, Nagashima K, Nishikawa T, Kimura S, Fujiwara K, Rojkind M, Ogata I. HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins. Comp Hepatol. 2004 Jan 14;3 Suppl 1:S21-S23.
2. Lechuga CG, Hernandez-Nazara ZH, Rosales JA, Morris ER, Rincon AR, Rivas-Estilla AM, Esteban-Gamboa A, Rojkind M. Transforming Growth Factor- b 1 Modulates Matrix metalloproteinase-13 Expression in Hepatic Stellate Cells at the Transcriptional and Post-transcriptional Levels by Complex Mechanisms Involving p38MAPK, ERK1/2 and p70 S6K . Am. J. Physiol. 287: G974-G987, 2004.
3. Kannangai R, Diehl AM, Sicklick J, Rojkind M, Thomas D, Torbenson M. Hepatic angiomyolipoma and hepatic stellate cells share a similar gene expression profile. Hum Pathol.; 36:341-347, 2005
4. Svegliati-Baroni, G., Inagaki, Y., Rincon-Sanchez,AR., Else, C., Saccomanno, S.,Benedetti, A.,Ramirez, F., and Rojkind, M. Early response of a 2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF- b 1 independent. Hepatology, 2005; 42:343- 352.
5. Sicklick JK, Li YX, Choi SS, Qi Y, Chen W, Bustamante M, Huang J, Zdanowicz M, Camp T, Torbenson MS, Rojkind M, Diehl AM. Role for hedgehog signaling in hepatic stellate cell activation and viability. Lab Invest. 2005 Nov;85(11):1368-80.
6. Sicklick JK, Choi SS, Bustamante M, McCall SJ, Hernandez Perez E, Huang J, Li YX, Rojkind M, Diehl AM. Evidence for Epithelial-Mesenchymal Transitions in Adult Liver Cells. Am J Physiol Gastrointest Liver Physiol. 291: 291: G575-G583, 2006.
7. Zhang Y, Ikegami T, Honda A, Miyazaki T, Bouscarel B, Rojkind M, Hyodo I, Matsuzaki Y. Involvement of integrin-linked kinase in carbon tetrachloride-induced hepatic fibrosis in rats., Hepatology. 2006; 44:612-22.
8. Lechuga CG, Hernandez-Nazara ZH, Hernandez E, Bustamante M, Desierto G, Cotty A, Dharker N, Choe M, Rojkind M. PI3 Kinase is involved in PDGF-{beta} receptor up-regulation post-PDGF-BB treatment in mouse hepatic Stellate cells. Am J Physiol Gastrointest Liver Physiol. 291: G1051-G1061, 2006.
9. Nieto, N and Rojkind, M: Repeated whiskey binges promote liver injury in rats fed a choline-deficient diet J. Hepatol. 46: 330-339, 2007.