Raphael Zidovetzki

The research in this laboratory follows three main topics.

1) Membrane Biophysics. Role of lipid bilayer structure in the mechanism of activation of protein kinase C.
A long-standing focus of research in my laboratory involves elucidation of the details of the regulation of membrane-associated enzymes, specifically phospholipase A2 and protein kinase C (PK-C) by physicochemical parameters of the lipid bilayer structure. Protein kinase C represents at present the paradigm for enzymes regulated by the structure of the lipid bilayer membrane. Membrane lipids, form a variety of different lyotropic liquid crystalline structures, e.g. lamellar (bilayer) and various nonbilayer structures, such as inverted hexagonal and cubic. Almost every membrane studied so far contains at least one lipid that by itself in water forms a nonbilayer lipid phase. This leads to very important consequences for the physicochemical properties of the biological membrane and for membrane function, especially taking into account that most reactions in living cells take place not in solution, but on membrane surfaces.

In case of PK-C, current models of activation for this enzyme involve the association of inactive cytosolic proteins with membranes containing acidic phospholipids in a Ca²⁺-dependent manner, which causes a conformational change in the protein and its association with the lipid membrane. The association is strongly facilitated by the presence of phorbol ester or the natural co- factor 1,2-sn-diacylglycerol (DAG) which is produced as a result of stimulus-generated activation of phospholipases C or D. PK-C activation is also modulated by the structure of the lipid bilayer, specifically as a result of stress, or destabilization of the lipid bilayer which is caused by the presence of nonbilayer-forming lipids, such as DAG.

We provided a detailed characterization of the effects of DAGs on lipid bilayer structure and PK-C activity. We have established that PK-C is strongly activated by specific types of perturbations in lipid bilayer structure, including increased tendency to form nonbilayer lipid phases and formation of microdomains enriched in acidic phospholipid, phosphatidylserine (PS).These results provide strong support to the hypothesis that the physicochemical parameters of the lipid membranes, and specifically the tendency to form nonbilayer phases, play an important role in the mechanism of PK-C activation. Thus, ceramides, DAGs, and unesterified fatty acids, with their diverse biological effects, can modulate PK-C activity by perturbing lipid bilayer structure and providing a potential crosstalk between the respective signal transduction pathways. The PK-C- activating synergism observed in the effects of DAG/unesterified fatty acid and DAG/ceramide can be utilized as a coincidence detector, efficiently activating PK-C with sub-optimal concentrations of these second messengers.

The results also have broader implications. Because many cellular membranes exist under conditions of high curvature stress, being close to the hexagonal phase transition, the physicochemical properties of these membranes are expected to be sensitive to changes in DAG and ceramide concentrations, as well as other membrane components and lipophilic agents which may modulate curvature stress. Therefore, membrane-associated enzymes that are sensitive to curvature strain would be highly responsive to changes in DAG and ceramide concentrations. It follows that a broad range of cellular activities may be modulated by the biophysical characteristics of the plasma membrane, which is a function of lipid composition as determined by dietary fat intake and the activity of lipid metabolic enzymes. Moreover, lateral heterogeneity of biological membranes allows concurrent diversity in the signal processing properties of specific cell surface locations, allowing membrane regions to function as discrete units with a capacity to act individually upon multiple signaling inputs. This hypothesis adds a new dimension to our conceptual understanding of the process of transmembrane signal transduction.

2) Intracellular Signal Transduction. Mechanisms of activation of human brain endothelial cells.
The overall goal is to identify the intracellular signal transduction pathways utilized by: a) stroke risk factors in the pathological activation of human cerebral endothelial cells. The hypothesis is that characterization of these pathways will lead to development of novel therapies to prevent or reduce the risk of ischemic stroke, and b) elucidate details of endothelial cell activation leading to angiogenesis in order to develop novel anti-angiogenesis therapies against malignant brain tumors.This medical-oriented research is conducted using primary patient-derived human brain endothelial cells, and proceeds in two main directions.
     a) Activation of human brain endothelial cells by the stroke risk factor: endothelin 1 (Et-1). Inflammatory cells, particularly neutrophils and macrophages, are associated with increased brain tissue damage following ischemic stroke, during reperfusion. The accumulation of leukocytes is due to increased production of chemotactic factors for these leukocytes. We have shown that Et-1 induces human brain endothelial cells to produce IL-8, establishing a connection between activation of cerebral endothelial cells and post-ischemic tissue damage caused by accumulation of neutrophils. We have also characterized the signal transduction pathways involved in this process, starting with the involvement of a specific class of Et-1 receptor, ETA, followed by subsequent activation of PK-C, protein tyrosine kinase, and transcription factor NF-B. Blocking this process at any step by receptor antagonists or specific kinase inhibitors completely abolished the Et-1-induced IL-8 production, suggesting novel therapeutic targets for reducing stroke-associated tissue damage.
     b). Development of novel anti-angiogenic anti-cancer therapies based on selective inhibition of signal transduction pathways. Angiogenesis, the growth of new blood vessels from preexisting vessels, is a complex process initiated by vascular destabilization followed by endothelial cell migration, proliferation, and eventually tubule formation. Angiogenesis is essential for cancer progression as demonstrated by the correlation between microvessel density and poor clinical outcome. Therefore understanding the mechanisms of angiogenesis in cancer is likely to lead to more effective cancer therapy. We investigated the mechanism of Et-1-induced migration of primary cultures of human brain endothelial cells. A dissection of the signal transduction pathways involved in this process demonstrated the participation of both Et-1 receptor types, and of multiple signal transduction enzymes, including Src, Ras, PK-C, PK-A, ERK, p38, and JNK. This dependence of angiogenesis on a complex signaling system provides an opportunity for a therapeutic intervention at various regulation sites.

3) Bioinformatics. Development of novel statistical analysis of gene microarrays and application to studying human diseases.
We are developing and using novel statistical analysis of gene microarray data, and have recently applied them to the study of cellular changes in the brains of Alzheimer patients. We have also developed software to design the chips and to analyze the data in the study directed to search for genes associated with Systemic Lupus Erythematosus.


Representative Publications:

• Milan, J., Charalambous, C., Elhag, R., Chen, T. C., Li, W., Guan, S., Hofman, F. M., Zidovetzki, R. (2006) Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration. Am. J. Physiol. Cell. Physiol., 291:C155-64.
• Wu, Z., Guo, H., Chow, N., Sallstrom, J., Bell, R. D., Deane, R., Brooks, A. I., Kanagala, S., Rubio, A., Sagare, A., Liu, D., Li, F., Armstrong, D., Gasiewicz, T., Zidovetzki, R., Song, X., Hofman, F., Zlokovic, B. V. (2005) Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Medicine, 11:959-65.
• Zidovetzki, R., Rost, B., Armstrong, D. L., Pecht, I. (2003) Transmembrane domains in the functions of Fc receptors. Biophys Chem., 100:555-75.
• Armstrong, D. L., Borchardt, D. B., Zidovetzki, R. (2002) Synergistic perturbation of phosphatidylcholine/sphingomyelin bilayers by diacylglycerol and cholesterol. Biochem. Biophys. Res. Commun., 296:806-12.
• Huang, H.-W., Goldberg, E. M., Zidovetzki, R. (1999) Ceramides perturb lipid bilayer structure and activate protein kinase C. Biophys J., 77: 1489-1497.
• Zidovetzki, R., Wang, J.-L., Kim, J. A., Chen, P., Fisher, M., Hofman, F.M. (1999) Endothelin-1 Enhances Plasminogen Activator Inhibitor-1 Production by Human Brain Endothelial Cells via Protein Kinase C-Dependent Pathway. Arteriosclerosis, Thrombosis & Vascular Biology 19: 1768-1775.
• Zidovetzki, R., Chen, P., Fisher, M., Hofman, F. M. (1999) Nicotine Increases Plasminogen Activator Inhibitor-1 Production by Human Brain Endothelial Cells via Protein Kinase C-Associated Pathway. Stroke. 30: 651-655.
• Goldberg, E. M., Zidovetzki, R. (1998) Synergistic effects of diacylglycerols and fatty acids on membrane structure and protein kinase C activity. Biochemistry 37: 5623-5632.
• Zidovetzki, R., Wang, J.-L., Chen, P., Jeyaseelan, R., Hofman, F. (1998) Human immunodeficiency virus tat protein induces interleukin-6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways. AIDS Res.Human Retroviruses 14: 873-878.
• Goldberg, E. M., Zidovetzki, R. (1997) Effects of dipalmitoylglycerol and fatty acids on membrane structure and protein kinase C activity. Biophys J. 73: 2603-2614.
• Zidovetzki, R. (1997) Membrane properties and the activation of protein kinase C and phospholipase A2. Current Topics in Membranes 44: 255-283.