We develop new biophysical and optical methodologies for biomedical research and clinical applications. Currently, we focus on developing technologies that (1) characterize early stages of disease and strategies for prevention of disease progression and (2) monitor responses to therapy in cancer and age-related macular degeneration (AMD). In collaboration with NCI and the Center for Information Technology (CIT), we have invented and are applying a new simple, robust, and high-throughput technology for automated, target-directed microtransfer to optimize the isolation of specific populations of cells and organelles from sections of complex tissues. This new technology is particularly suited to identifying critical but less abundantly expressed genes, proteins, and lipids. In our AMD prevention research, we have developed a novel biophysical model of chronic steady-state levels of the photoproduct A2E (N-retinylidene-N-retinylethanolamine) within the human macula that critically depends on the normal shifts in the spectral irradiance as the lens yellows with age. We have designed spectrally selective sunglasses that optimally reduce the two photochemical processes determining A2E levels. In collaboration with NEI, we are developing noninvasive clinical measurements of A2E to test our model and our ability to reduce A2E levels in individuals at risk for macular pathology that, we believe, is induced by elevated A2E levels.
Laser microdissection and molecular diagnostics technology development
Bonner; in collaboration with Emmert-Buck, Mage, Pohida, Sackett
Integrative molecular biology requires an understanding of the interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. A fundamental argument for laser capture microdissection (LCM) of tissues is that, without separation of specific cell populations from complex tissues, we will miss critical control functions of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. Without detecting changes in many of these critical effectors, the integrative understanding of tissue function and pathology will not advance. In complex tissues, particularly among pathological variations, it is exceptionally difficult to measure the majority of molecules that are present at low copy number per cell without first isolating specific cell populations. For example, among partially sequenced cDNA libraries of the Cancer Genome Anatomy Project, only LCM-dissected ovarian cancer cDNA libraries are exceptionally informative about ovarian cancer biology. After LCM isolation of pure target cells, the library construction protocol selectively amplified a small number of rarer transcripts to a level that allowed statistical comparison of their expression between highly purified cells of low- and high-malignancy potential. Many of these “overamplified” genes, which are expressed at higher levels in the high-malignancy potential than in the low-malignancy potential ovarian cancer libraries, are known oncogenes and genes associated with invasion and metastatic processes in other tissues.
The LCM techniques that we started developing 10 years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells of complex tissues. However, in global proteomic and lipid studies without molecular amplification methods, the quantity of isolated cells sufficient to perform accurate characterization of less abundant species is problematic, given that the microscopic visualization, targeting, and isolation in laser microdissection has a maximal rate of about one to 20 cells per second, depending on the cells’ microscopic distribution within the tissues. In collaboration with NCI and CIT, we recently invented and are now refining an automatic “target-directed microtransfer” technique based on macromolecule-specific labeling of cells. The technique (patent pending) does not require user visualization or microscopic targeting and substantially increases throughput rates. It is built on our solid physical understanding of thermoplastic microtransfer and uses a much simpler device and transfer films than commercial laser microdissection instruments. Our current prototype is capable of isolating all specifically immunolabeled cells or organelles within a 1-cm2 region of a standard immunostained tissue section in about 5 seconds, which corresponds to specific separation from approximately 50,000 cells per second. With this technique, we can exceed the cell separation rates of standard technologies such as fluorescence-activated cell sorting while preserving our ability to harvest directly from standard sections of complex tissues. This rapid, automated microtransfer method has improved spatial resolution (about 1 micron) and, consequently, is particularly well suited to isolating highly dispersed, specific cell populations (e.g., stem cells or only those neurons in the supra-optic nucleus that express vasopressin) or specific organelles (e.g., neuronal nuclei in the brain). The transfer film preserves the spatial relationships (morphology) among the specific cells in the tissue. As the evolving technology becomes more robust, we will seek to integrate the microtransfer with molecular profiling of specific cells within tissues, including routine proteomic and lipidomic analyses, particularly for the large number of less abundant molecular species.
Celeste A, Fernandez-Capetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A, Bonner RF, Bonner WM, Nussenzweig A. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 2003;5:675-679.
Jones MB, Michener CM, Blanchette JO, Kuznetsov VA, Raffeld M, Serrero G, Emmert-Buck MR, Petricoin EF, Krizman DB, Liotta LA, Kohn EC. The granulin-epithelin precursor/PC-cell-derived growth factor is a growth factor for epithelial ovarian cancer. Clin Cancer Res 2003;9:44-51.
Mage R, Bonner RF, Obiakor HT. Microdissection of single or small numbers of cells for analyses of DNA and RNA by PCR. In: Weissensteiner T, Griffin HG, Griffin A, eds. PCR Technology: Current Innovations, 2nd edition. Boca Raton, FL: CRC Press, 2003;29-36.
Tangrea MA, Chuaqui RF, Gillespie JW, Pohida TJ, Bonner RF, Emmert-Buck MR. Expression microdissection: operator-independent retrieval of cells for molecular profiling. Diagn Mol Pathol 2004;13:207-212.
UNITED STATES PATENTS
#6,569,639 May 27, 2003: Liotta LA, Buck MF, Weiss RA, Zhuang Z, Bonner RF. Isolation of cellular material under microscopic visualization.
#6,420,132 July 16, 2002: Bonner RF, Goldstein SR, Smith PD, Pohida TJ. Precision laser capture microdissection utilizing short pulse length.
#6,251,516 June 26, 2001: Bonner RF, Liotta L, Buck M, Krizman DB, Chuaqui R, Linehan WM, Trent JM, Goldstein SR, Smith PD, Peterson JI. Isolation of cellular material under microscopic visualization.
Patent pending: Bonner RF, Pohida T, Buck M, Tangrea M, Chuaqui R. Target-directed microtransfer.
Gene expression during normal development and pathology progression
Bonner; in collaboration with Boukari, Emmert-Buck, Sackett
If microdissection and molecular analysis can be made clinically practical, the expression levels of sets of approximately 20 to 100 critical, stage-specific disease markers within a selected cell population might provide reliable diagnosis and intermediate endpoints of response to molecular therapies in individual patients. Our analysis of large gene expression and protein databases suggests that a significant fraction of all genes is expressed in any specific cell type and that the levels of gene products universally exhibit a highly skewed power-law distribution similar to those characterizing many other complex systems (Kuznetsov, Genetics 2002;161:1321; Kuznetsov, J Biol Syst 2002;10:381). We have developed mathematical models for the evolution of such distributions that predict the observed distributions of genes, protein domains, and gene expression observed in species of increasing biological complexity.
To permit more routine and simpler multiplex molecular diagnostics, we are attempting to develop new approaches for better integration of our thermoplastic microtransfer methods of microdissection with downstream macromolecular analysis. A key feature is the use of the polymer matrix in which target cells are embedded for affinity purification and then for direct optical detection within the transparent polymer. We are using a variety of microscopy techniques in our laboratory to characterize quantitatively protocols for incorporating affinity nanoparticles in the tissue and polymer matrix. Over the longer term, we foresee the use of in situ optical labels to quantify the spatial distributions of specific molecules captured within the microtransfer and retained following simple purification steps. Coupling the robust and simple automatic microdissection with rapid purification and detection of species might provide unique abilities to follow macromolecular changes in normal tissue development as well as in pathologies such as cancer progression within prostate, colon, breast, lung, and ovary tissues. In continuing collaborations with NCI, we have developed standard procedures for the isolation of normal and pathological cells from clinical specimens. We have used our models of the statistics of expression levels in cell populations to identify genes differentially expressed in cancer progression. To date, our analysis points to a critical role for many less abundantly expressed genes at a critical stage of ovarian cancer progression, suggesting that, for most cancers, diagnostic marker sets should include such low-abundance transcripts. Such a notion is guiding our research in statistics of less abundant gene products and suitable detection methods.
We foresee an evolution of molecular diagnosis from one based on qualitative or quantitative analysis of a few key macromolecules to one in which special clustering algorithms analyze complex multivariate databases. Such analyses should permit a more complete identification of highly correlated clinical cases and allow us to characterize their response to molecular therapies specifically designed to prevent progression.
Kuznetsov VA. Family of skewed distributions associated with the gene expression and proteome evolution. Signal Processing 2003;83:889-910.
Mutsuga N, Shahar T, Verbalis JG, Brownstein MJ, Xiang CC, Bonner RF, Gainer H. Selective gene expression in magnocellular neurons in rat supraoptic nucleus. J Neurosci 2004;24:7174-7185.
Prevention of progression of age-related macular degeneration through photoprotection
Meyers, Ostrovsky, Kisseleva, Bonner; in collaboration with Avetisov, Caruso, Chew, de Monasterio, Ferris, Sieving
The associations of age-related macular degeneration (AMD) with cataracts, earlier cataract surgery, cumulative exposure to sunlight, and pigmentation all support the hypothesis that chronic photochemical injury drives macular changes with age and AMD progression. With age, lipofuscin accumulates in the retinal pigment epithelium (RPE) and co-localizes with acute photosensitization of reactive oxygen intermediates (ROI) in the primate retina. Lipofuscin granules contain at least 10 fluorescent photochemical products, includng A2E (N-retinylidene-N-retinylethanolamine), its epoxides, and other as yet chemically unidentified A2E-related fluorophores. The precursors of these fluorophores originate from reactions of all-trans-retinal within rod outer segment (ROS) discs during periods associated with significant rhodopsin bleaching (i.e., normal daylight). Although RPE lysosomal processing digests over 99 percent of the shed ROS contents, A2E and related fluorophores are not digested but instead concentrate in lipofuscin granules. By age 60, the average concentration of A2E within RPE cells reaches around 400 micromolar in normal eyes. However, A2E is toxic to cellular membranes at much lower concentrations. We hypothesize that segregation of A2E into lipofuscin granules and prevention of its redistribution into critical membranes is required for RPE health.
We sought to model both the normal age-related accumulation of potentially damaging photoproducts in the RPE and the changes induced by external spectral filtering of light reaching the macula. We developed a biophysical model that uses normal values of pupil size, lens transmission, and rod dark adaptation to determine average retinal spectral irradiance and resulting production of A2E-related species in the ROS and RPE as a function of age and ambient light intensity. Our model predicts a decline of about one-third in the action spectra–weighted short-wavelength macular irradiance with each decade of life and a nearly constant production rate of A2E-related fluorophores in the RPE during the first 60 years of life (falling significantly thereafter). A similar age dependence of total lipofuscin granule volume and total fluorescence per RPE cell has been reported in human cadaver eyes. With age, the rate of lipofuscin increase is slower than the rate of decrease in short-wavelength macular irradiance in the phakic eye; consequently, ROI photosensitization in the RPE should also fall with increasing age. Photo-oxidative stress in the outer retina might arise from the smaller amounts of A2E-related fluorophores observed in critical membranes of the RPE/BM (Bruch's Membrane) complex. However, if the RPE/BM complex were the site of photo-oxidative injury driving AMD progression, the magnitude and rate of oxidative injury would be expected to increase dramatically following cataract removal and intraocular lens (IOL) implantation.
Consequently, we propose a novel hypothesis that photochemically induced singlet oxygen generation within RPE lipofuscin granules induces the chemical alteration of accumulating A2E, thereby limiting the steady-state levels of A2E ([A2E]ss) in the RPE, the redistribution of A2E into retinal membranes, and A2E chemical toxicity. Singlet oxygen reacts with its A2E to form A2E epoxides that then react to form increasingly complex cross-linked molecules. As short-wavelength macular irradiance falls with age, the rate of A2E photo-oxidation falls up to 20-fold, causing [A2E]ss in the normal phakic eye to increase even as rod bleaching and A2E production decrease. Our theoretical model of macular aging reproduces the normal age dependence of lipofuscin and A2E and provides a primary cytotoxic mechanism in which, once A2E reaches a threshold concentration in the RPE cell, A2E redistribution into critical membranes causes damage with or without additional photo-activation. The model also predicts that, in normal eyes, nearly constant levels of A2E are maintained at a given age and lens color irrespective of total ambient light exposure. It is primarily the yellowing of the lens with age that distorts the original spectral balance between rate of production and rate of photo-oxidation found in youth, allowing the [A2E]ss to rise with age. If our model is correct, then restoring or optimizing the spectral balance by wearing spectrally selective sunglasses could significantly lower A2E levels and may prevent associated macular degenerations.
Noninvasive, quantitative imaging of retinal autofluorescence associated with A2E levels could permit clinical validation of our predictions of both the photochemical changes associated with lens status and the benefits of specific spectral photoprotective filters. We have designed vermilion sunglasses that should optimally reduce both rod activation in bright ambient light and the accumulation of toxic photoproducts in the RPE. In collaboration with NEI and the Eye Institute of the Russian Academy of Medical Sciences, we are designing clinical studies of the effects of such filters on A2E levels in the RPE and on progression of both early and moderate AMD following cataract surgery and IOL implantation and in young patients with Stargardt’s macular dystrophy.
Meyers SM, Ostrovsky MA, Bonner RF. A model of spectral filtering to reduce photochemical damage in age-related macular degeneration. Trans Am Ophthal Soc 2004;102:83-95.
1Russian Academy of Sciences, Moscow, Russia
COLLABORATORS
Sergei Avetisov, MD, Eye Institute of the Russian Academy of Medical Sciences, Moscow, Russia
Hacene Boukari, PhD, Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, MD
Rafael Caruso, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Emily Chew, MD, Division of Epidemiology and Clinical Research, NEI, Bethesda, MD
Francisco de Monasterio, MD, PhD, Office of the Clinical Director, NEI, Bethesda, MD
Michael R. Emmert-Buck, MD, PhD, Laboratory of Pathology, NCI, Bethesda, MD
Frederick Ferris, MD, Office of the Clinical Director, NEI, Bethesda, MD
Rose G. Mage, PhD, Laboratory of Immunology, NIAID, Bethesda, MD
Sanford Meyers, MD, Retina Consultants, Des Plaines, IL
Tom Pohida, MSEE, Center for Information Technology, NIH, Bethesda, MD
Dan Sackett, PhD, Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, MD
Paul Sieving, MD, PhD, Director, NEI, Bethesda, MD
For further information, contact bonnerr@mail.nih.gov.