MEDICAL BIOPHYSICS
, Head, Section on Medical Biophysics
Zigurts Majumdar, PhD, Research Fellow
Eleanor Ory, BSc, Postbaccalaureate Fellow
Tatiana Kisseleva, MD, Guest Researcher
Sanford Meyers, MD, Guest Researcher1
Mikhail Ostrovsky, PhD, DSc, Guest Researcher2
Currently, we are developing optical technologies to characterize early stages of disease and to monitor responses to therapy in cancer and age-related macular degeneration (AMD). We have invented and are applying a new simple, robust, high-throughput technology for automated, target-directed microtransfer to optimize the isolation of specific populations of cells and organelles from sections of complex tissues, enabling studies of less abundantly expressed genes, proteins, and lipids. Our AMD research has created a novel biophysical model that predicts that A2E (N-retinylidene-N-retinylethanolamine) levels increase with age owing to changes in retinal spectral irradiance. We have designed spectrally selective, bicolored sunglasses to reduce the photochemical processes producing A2E in the photoreceptors and/or A2E’s photo-oxidation in the retinal pigment epithelium (RPE). Our noninvasive imaging of A2E and related fluorophores in clinical studies is designed to test our model and ability to alter A2E levels in individuals at risk for AMD. We are further developing imaging technologies suitable for characterizing changes in microscopic early macular pathology that may permit improved studies of the effectiveness of a wide range of interventions designed to reduce or prevent AMD.
Laser microdissection and molecular diagnostics technology development
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. In complex tissues—particularly among pathological variations—it is exceptionally difficult to measure the majority of molecules that are at low copy number per cell without first isolating specific cell populations. With our newest inventions, we are increasing the microdissection resolution to the organelle level.
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 in 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 as microscopic visualization, targeting, and isolation in laser microdissection has a maximal rate of 1 to 20 cells per second depending on the cells’ microscopic distribution within the tissues. Recently, in collaboration with the NCI and CIT, we developed and are now refining an automatic “target-directed microtransfer” technique based on macromolecule-specific labeling of cells, a technique that does not require user visualization or microscopic targeting and that is capable of much higher throughput rates. This technique (patent pending) is built on our solid physical understanding of thermoplastic microtransfer and uses a much simpler device and transfer films than those associated with 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, thus corresponding to specific separation from approximately 100,000 cells per second. With this technique, we can exceed the cell separation rates of standard technologies such as fluorescence-activated cell sorting but preserve our ability to harvest directly from standard sections of complex tissues. This rapid, automated microtransfer method offers improved spatial resolution (about 1 µ) and consequently is particularly well suited to isolating highly dispersed, specific cell populations (such as stem cells or only those neurons in the supra-optic nucleus that express vasopressin) or specific organelles (such as neuronal nuclei in the brain). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. As this technology becomes more robust, we will integrate 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 and their post-translational modifications.
Wang H, Owens JD, Shih JH, Li M-C, Bonner RF, Mushinski JF. Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA. BMC Genomics 2006;7:97-105.
United States Patents
#6,569,639 issued May 27, 2003: Isolation of cellular material under microscopic visualization. Liotta LA, Buck MF, Weiss RA, Zhuang Z, Bonner RF.
#6,420,132 issued July 16, 2002: Precision laser capture microdissection utilizing short pulse length. Bonner RF, Goldstein SR, Smith PD, Pohida TJ.
#6,251,516 issued June 26, 2001: Isolation of cellular material under microscopic visualization. Bonner RF, Liotta L, Buck M, Krizman DB, Chuaqui R, Linehan WM, Trent JM, Goldstein SR, Smith PD, Peterson JI.
Patent pending: Target-directed microtransfer. Bonner RF, Pohida T, Buck M, Tangrea M, Chuaqui R.
Gene expression during normal development and pathology progression
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 VA, Signal Processing 2003;83:889). 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 (Kuznetsov et al., Genetics 2002;161:1321).
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 of our approach is the use of the polymer matrix transfer film in ways that facilitate subsequent macromolecular analyses. We are using a variety of microscopy techniques to characterize quantitatively Laser microcapture and polymer matrix–cell macromolecule interactions. For the long term, we foresee integration of the transfer films within the downstream molecular analysis by, for example, using 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 for following macromolecular changes in normal tissue development and in pathologies such as cancer progression and its response to therapy. In continuing collaborations with the NCI, we have developed standard procedures for isolating 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 specific targets within complex tissues. To date, our analysis points to a critical role for many less abundantly expressed genes at a critical stage of ovarian cancer progression and suggests that, for most cancers, critical diagnostic marker sets should include such low-abundance transcripts. This notion is guiding our research in statistics of less abundant gene products and suitable isolation and coupled molecular analysis 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.
Grover AC, Tangrea MA, Woodson KG, Wallis BS, Hanson JC, Chuaqui RF, Gillespie JW, Erickson HS, Bonner RF, Pohida TJ, Emmert-Buck MR, Libutti SK. Tumor-associated endothelial cells display GSTP1 and RARb2 promoter methylation in human prostate cancer. J Transl Med 2006;4;13-9.
Prevention of progression of age-related macular degeneration through photoprotection
Our clinical studies are designed to test our biophysical model and clinical hypothesis that spectral imbalances of light reaching the retina lead to increased levels of toxic photochemicals within the retinal pigment epithelium, thereby driving the early stages of AMD. If our hypothesis is correct, we believe that, during the earliest stages of retinal pathology, this process may be reversible. Therefore, progression to more advanced AMD might be prevented by wearing external spectral sunglasses or otherwise altering the spectrum of the ambient light reaching the retina. We have designed such sunglasses and have begun clinical studies in pseudophakes to attempt to image induced changes noninvasively in the distribution of the toxic photochemicals within the human retina. In addition, we are seeking to improve spectral autofluorescence imaging of the retina in order to provide high-resolution, molecularly specific images of early microscopic lesions, thereby permitting us to follow the lesions’ changes over time in relation to A2E levels. In collaboration with computational image analysis experts at University of Maryland, we are seeking to develop new quantitative tools for following noninvasively the earliest changes in macular pathology in AMD and other retinal diseases. We hope to provide better early diagnosis and ways to monitor early intervention and disease prevention therapies.
In our clinical collaborations at the NIH and with the Eye Diseases Institute of the Russian Academy of Medical Sciences, we are seeking to develop noninvasive spectral imaging methods to study early photochemical injury and the means of limiting it. Lipofuscin accumulates with age in the RPE and colocalizes with acute photosensitization of reactive oxygen intermediates (ROIs) in the primate retina. The associations of AMD with cataracts, earlier cataract surgery, cumulative exposure to sunlight, and pigmentation support hypotheses that chronic photochemical injury drives macular changes with age and AMD progression. We have developed a biophysical model of (1) the normal accumulation during aging of potentially damaging photoproducts in both the RPE and Bruch’s Membrane complex and (2) changes induced by additional spectral filtering of light reaching the macula. The precursors (A2PE and A2PEH2) of fluorophores that accumulate within RPE lipofuscin granules originate from reactions of all-trans-retinal within the receptor outer segments (ROSs) during periods associated with normal daylight. Although RPE lysosomal processing enzymatically digests over 99 percent of shed ROS contents, A2E and related fluorophores are not digested but rather concentrate into lipofuscin granules. By age 60, the average concentration of A2E within RPE cells reaches 400 µM 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 A2E’s redistribution into critical membranes is required for RPE health.
Our novel hypothesis posits that photochemically induced singlet oxygen generation within RPE lipofuscin granules limits steady-state levels of A2E ([A2E]ss) in the RPE, A2E redistribution into retinal membranes, and associated A2E chemical toxicity. As short-wavelength macular irradiance falls with age, the rate of A2E photo-oxidation declines by 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 (Meyers et al., Trans Am Ophthalmol Soc 2004;102:83) reproduces the normal age dependence of lipofuscin and A2E. It 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 normal eyes maintain nearly constant levels of A2E 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 the rate of production and the rate of photo-oxidation found in youth, allowing [A2E]ss to rise with age. If our model is correct, then restoring or optimizing the spectral balance by wearing external spectrally selective sunglasses could significantly lower A2E levels and may prevent associated macular degenerations. Our clinical studies using noninvasive, quantitative imaging of retinal autofluorescence associated with A2E levels are designed to test our hypotheses and the benefits of specific spectral photoprotective filters.
1 Retina Consultants, Des Plaines, IL
2 Institute of Biochemical Physics, Russian Academy of Sciences
COLLABORATORS
Sergei Avetisov, MD, Eye Institute of the Russian Academy of Medical Sciences, Moscow, Russia
David Berler, MD, Washington Eye Physicians and Surgeons, Chevy Chase, MD
Emily Chew, MD, Clinical Branch, NEI, Bethesda, MD
Denise Cunningham, CRA, RBP, MEd, Clinical Branch, NEI, Bethesda, MD
Francisco de Monasterio, MD, PhD, Clinical Branch, NEI, Bethesda, MD
Michael R. Emmert-Buck, MD, PhD, Laboratory of Pathology, NCI, Bethesda MD
Paul Meltzer, MD, PhD, Cancer Genetics Branch, NCI, Bethesda, MD
Tom Pohida, MSEE, Computational Biology and Electronics Laboratory, CIT, NIH, Bethesda, MD
Jaime Rodriguez-Canales, MD, Surgery Branch, NCI, Bethesda, MD
Dan Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
For further information, contact bonnerr@mail.nih.gov
