Faculty, Graduate Program
Membrane Protein Topogenesis and Ocular Angiogenesis
Our laboratory is studying (1) the mechanism whereby proteins are asymmetrically integrated into cell membranes and (2) the role of integrins and integrin antagonists in ocular angiogenesis.
Topogenesis of Rhodopsin
Polytopic membrane proteins span the lipid bilayer several times and have hydrophilic domains exposed on alternate sides of the membrane. Opsin (the apoprotein of rhodopsin) is representative of the larger family of G-protein coupled receptors that have seven transmembrane segments (TMS) and eight hydrophilic domains, four of which face the biosynthetic compartment of the cell and four of which are extracellular. By constructing a series of opsin mutants, each containing only a single TMS, we were able to demonstrate that opsin has at least 5 internal signal sequences each of which also expresses a strong or weak stop--transfer sequence. We have recently extended these studies to examine how these topogenic sequences may function to sequentially insert the entire protein into the membrane. In collaboration with Sanford Simon's group at Rockefeller, we have shown that opsin, within a range of nascent peptide lengths, targets and translocates equally efficiently co- and post-translationally and that SRP is required for both co- and post-translational targeting. Futhermore, we have demonstrated that this post-translational targeting and translocation requires nucleotide triphosphates (GTP alone is sufficient to fully restore targeting), but not cytosolic proteins. The addition of ATP was not specifically required and non-hydrolyzable analogs of ATP that blocked 90% of the ATPase activity also had no inhibitory effect on translocation.
Topology of the Na+/Ca++ exchanger from cardiac muscle and photoreceptors.
In collaboration with Ken Philipson's group we have been investigating the topology of the cardiac Na+--Ca++ exchanger. Based on hydropathy analysis of the amino acid sequence, the exchanger is proposed to contain 12 hydrophobic segments, the first of which is a cleaved signal sequence. By using a variety of reporter domains (glycosylation sites, epitopes and proteolytic cleavage sites), we have begun to analyze the topology of the exchanger both in vitro and in oocyte expression systems. A full length cDNA clone from photoreceptors has also been obtained and is being similarly analyzed.
The cardiac exchangers have a cleaved amino--terminal signal sequence. Since nearly all other polytopic eucaryotic membrane proteins do not have cleaved signal sequences, we are investigating the putative role of such a sequence in the insertion and targeting of these exchangers. Our results demonstrate that the native, cleaved amino terminal signal sequence is not necessary for insertion of a functional exchanger into the cell membrane. In contrast, the photoreceptor exchanger does not have a cleaved amino terminal signal sequence. If the N-terminal 65 amino acids are deleted, translocation of the N-terminus of the protein is disrupted, but the remainder of the exchanger is integrated into the membrane. Functionally expressed exchanger is being studied using ion exchange assays and two photon scanning laser confocal microscopy of live cell cultures and retinal explants.
The vast majority of diseases that cause catastrophic loss of vision do so as a result of abnormal blood vessel growth. Pathological retinal or choroidal neovascularization lead to visual loss in diabetic retinopathy (DR) and age related macular degeneration (ARMD), respectively. Similarly, tumors depend on a blood supply for their growth and use these new vessels as an avenue for metastasis. Blood vessels themselves can generate tumors (e.g., hemangiomas) when the growth and organization of vascular endothelial cells is not properly controlled. We are studying each of these areas with the goal of understanding mechanisms of ocular neovascularization in normal and pathological situations. We have used a neonatal mouse retina model to identify regulators of developmental angiogenesis and understand endothelial guidance mechanisms as well as, in a long-standing collaboration with the Cheresh laboratory, evaluate the role of integrins in this process. In other studies, we have shown that bone marrow-derived endothelial precursor cells (EPCs) specifically target retinal astrocytes, incorporate into new vessels and, in a model for retinal degeneration, rescue and stabilize a degenerating retinal vasculature. In collaboration with the Schimmel laboratory we have found that these cells can also be transfected with a plasmid encoding a secreted form of a newly discovered anti-angiogenic, T2 (a fragment of tryptophan tRNA synthetase). Injection of these cells into newborn mouse eyes results in significantly reduced retinal vascularization. Most recently, we have demonstrated that EPCs have a profound neurotrophic effect when injected into eyes of mice with inherited retinal degeneration; not only is the vasculature rescued in these mice, but photoreceptors and visual function are also preserved.
Glioblastoma multiforme is an incurable brain tumor that is usually fatal within one year. We are using gene therapy and a rat model of this disease to study the efficacy of an anti-angiogenic approach in treating these tumors. Hemangiomas are endothelial tumors that proliferate rapidly and later involute spontaneously. We are using DNA microarrays to study changes in gene expression as these tumors progress with the goal of identifying new targets for therapy for these tumors and identifying novel regulators of angiogenesis. In a collaboration with the Nemerow laboratory, we have used pseudotyped adenovirus to selectively target specific cell types in the retina; using the appropriate fiber type we can deliver transgenes to cells, such as photoreceptors, that ordinarily are not targeted by adenovirus.
M.D., Medicine, State University of New York Downstate Medical Center, 1983
Ph.D., The University of Chicago, 1976
Member, Visual Sciences C Study Section, National Eye Institute; Consultant, Genetics of Diabetic Retinopathy Workshop, National Eye Institute.
For a complete list of publications: http://www.scripps.edu/friedlander/publications.php
Marchetti, V., Krohne, T.U., Friedlander, D.F., and Friedlander, M. (2010). Stemming vision loss with stem cells. J Clin Invest. Sep 1;120(9):3012-21. PMCID: PMC2929728.
Weidemann, A., Krohne, T.U., Aguilar, E., Kurihara, T., Takeda, N., Dorrell, M.I., Celeste Simon, M., Haase, V.H., Friedlander, M., Johnson, R.S. (2010). Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina. Glia. Aug;58(10):1177-85. PMCID: PMC2993327.
Dorrell, M.I., Aguilar, E.A., Jacobson, R., Trauger, S., Friedlander, J.F., Suizdak, G. and Friedlander, M. (2010). Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy. Glia 58(1):43-54. PMCID: PMC2814838.
Dorrell, M.I., Aguilar, E.A., Jacobson, R., Yanes, O., Gariano, R., Heckenlively, J., Eyal Banin, E., Ramirez, E.G., Gasmi, M., Bird, A., Suizdak, G., and Friedlander, M. (2009). Treatment with antioxidants or neurotrophic factors preserves function in neurons damaged by neovascularization-associated oxidative stress. J. Clin. Invest. 119(3):611-623. PMCID: PMC2648679.
Scheppke, L., Aguilar, E., Gariano, R.F., Hood, J., Soll R., Yee, S., Noronha, G., Martin, M., Weis, S., Cheresh, D.A. and Friedlander, M. (2008). Retinal vascular permeability suppression by topical application of a novel VEGFR2/SRC kinase inhibitor in mice and rabbits J. Clin. Invest., 118(6):2337-46. PMCID: PMC2381746.
Dorrell, M., Aguilar, E., Scheppke, L., Barnett, F., and Friedlander, M. (2007). Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc. Natl. Acad. Sci. 104:967-972.
Ritter, M., Banin, E., Aguilar, E.A., Dorrell, M.I. Moreno S.K. and Friedlander, M. (2006). Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J. Clin. Invest. 116(12):3266-3276. PMCID: PMC1636693.
Banin, E., Dorrell, M.I., Aguilar, E., Ritter, M.R., Aderman, C.M., Smith, A.C.H., Friedlander, J., and Friedlander, M. (2006). T2-TrpRS inhibits pre-retinal neovascularization and enhances physiological vascular regrowth in oxygen-induced retinopathy as assessed by a new method of quantification. Invest. Ophthal. Vis. Sci., 47(5):2125-34.
Ritter, M., Reinisch, J., Friedlander, S.F., and Friedlander, M. (2006). Myeloid Cells in Infantile Hemangioma and a Possible Surrogate Model. Amer. J. Path. 168: 621-628. PMCID: PMC1606494.
Ritter, M., Aguilar, E., Banin, E., Scheppke, L., Uusitalo-Jarvinen, H. and Friedlander, M. (2005). Three Dimensional In Vivo Imaging of the Mouse Ocular Vasculature. Invest. Ophthal. Vis. Sci., 46:3021-26.
Otani, A., Kinder, K., Hanekamp, S., Nussinowitz,S., Heckenlively, J., and Friedlander, M. (2004). Neurotrophic rescue of retinal degeneration by intravitreally injected adult bone marrow derived lineage hematopoietic stem cells. J. Clinical Investigation, 114:765-774. PMCID: PMC516263.
Belting, M., Dorrell, M., Sandgren, S., Ahamad, J., Dorfleutner, D., Carmeliet, P., Mueller, B., Friedlander, M. and Ruf, W. (2004). Tissue factor signaling in angiogenesis. Nature Medicine 10:502-509.
Dorrell, M.I., Otani, A., Aguilar, E.A., Moreno, S. and Friedlander, M. (2004). Adult bone marrow-derived stem cells utilize R-cadherin to target sites of neovascularization in the developing retina. Blood 103: 3420-3427.
Dorrell, M.I., Aguilar, E. and Friedlander, M. (2004) Global gene expression analysis of the developing post-natal retina. Investigative Ophthal. Vis. Sci., 45:1009-19.
Ritter, M.R., Hanekamp, S., Dorrell, M.I., Rubens, J., Ney, J., Friedlander, D.F., Bergman, J., Cunningham, B.J., Eichenfield, L., Reinisch, J., Cohen, S., Veccione, T., Holmes, R., Friedlander, S.F., and Friedlander, M. (2004). Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis – A possible role for the immune system and IDO during involution. Lymphatic Research and Biology 1:291-299.
Otani, A., Slike, B., Dorrell, M. I., Hood, J., Kinder, K., Ewalt, K., Cheresh, D.A., Schimmel, P. and Friedlander, M. (2002). A fragment of human TrpRS as a potent antagonist of ocular angiogenesis. PNAS 99:178-183. PMCID: PMC117535.
Ritter, M., Dorrell, M., Edmonds, J., Friedlander, S., and Friedlander, M. (2002). IGF-2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis. PNAS 99:7455-7460.
Dorrell, M., Aguilar de Diaz, E., and Friedlander, M. (2002). Developmental vascularization of the retina is mediated by a pre-existing astrocytic template and specific R-cadherin adhesion. Investigative Ophthalmology and Visual Science 43:3500.
Otani, A., Kinder, K., Ewalt, K., Otero, F., Schimmel, P., Friedlander, M. (2002). Bone marrow derived stem cells cells target retinal astrocytes and have pro- or anti-angiogenic activity. Nature Medicine 8:1004-1010.
Friedlander, M., Brooks, P.C., Shaffer, R.W., Kincaid, C.M., Varner, J.A., Cheresh, D.A. (1995). Definition of two angiogenic pathways by distinct alpha v integrins. Science 270(5241):1500-2.