Robert F. Bonner, PhD, Head, Section on Medical Biophysics
Zigurts Majumdar, PhD, Research Fellow
Tatiana Kisseleva, MD, Guest Researcher
Sanford Meyers, MD, Guest Researcher
Mikhail Ostrovsky, PhD, DSc, Guest Researcher ¹

We develop new biophysical and optical methodologies for biomedical research and clinical applications. Currently, we focus on technologies for (1) characterizing early stages of disease and strategies for prevention of progression and (2) monitoring responses to therapy in cancer and age-related macular degeneration (AMD). We have invented and are applying a new simple, robust, and high-throughput technology for automated, target-directed microdissection 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 the human macula during aging in which chronic steady-state levels of the photoproduct A2E (N-retinylidene-N-retinylethanolamine) is predicted to increase with age due to red-shifts in spectral irradiance as the lens yellows with age. We have designed spectrally selective sunglasses that significantly reduce the two photochemical processes determining A2E levels. We hypothesize that elevated A2E levels induce macular pathology; therefore, we are developing noninvasive clinical imaging of A2E and related fluorophores to test our model and the ability of spectral filters to alter fluorophore levels in individuals at risk for macular degeneration.

Laser microdissection and molecular diagnostics technology development

Majumdar, Bonner; in collaboration with Emmert-Buck, 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, which we invented in collaboration with NCI and CIT, posits 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 proceed effectively. 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. 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 used by the project selectively amplified a small number of rarer transcripts to the level that allowed statistical comparison of their expression between highly purified cancer cells of low- and high-malignancy potential. Many of the "overamplified" genes that were more highly expressed in the high-malignancy potential rather than in the low-malignancy potential ovarian cancer libraries are known to be oncogenes and genes associated with invasion and metastatic processes in other tissues, though expressed at too low a copy number to be detected by library sequencing without microdissection and selective amplification.

The LCM techniques that we started developing 10 years ago use an infrared (IR) laser beam to focally melt an IR-absorbing thermoplastic polymer film in contact with a target cell within a complex tissue section visualized in a microscope. Although now widely used in molecular analysis of the genetics and gene expression changes within such specific target cells, LCM is more limited in global proteomic and lipid studies lacking a PCR-like amplification method because the quantity of isolated cells sufficient to perform accurate characterization of less abundant species is limited by a cell acquisition rate of about 1 to 20 cells per second. Recently, in collaboration with NCI and CIT, we invented an automatic "target-directed microtransfer" technique in which target-specific immunohistochemical stains selectively absorb the laser beam and locally cause a flexible thermoplastic film to pressurize so as to contact and microbond only with the stained targets as the beam is scanned rapidly over the entire sample. This technique (patent pending), which is capable of much higher throughput rates than its predecessor, uses a simpler device and does not require microscope use or time-consuming targeting decisions by the user. 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 100,000 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 μm) and consequently is particularly well suited to isolating highly dispersed, specific cell populations (e.g., stem cells or only neurons expressing vasopressin) or specific organelles (e.g., neuronal nuclei in the brain). As with LCM, spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. We are working toward better integration of the microtransfer with downstream 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.

United States Patents

Gene expression during normal development and pathology progression

MajumdarBonner; in collaboration with Emmert-Buck, Sackett,

If microdissection and molecular analysis can be made clinically practical, the expression levels 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. 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 simpler and more routine 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 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 to characterize quantitatively protocols for incorporating affinity nanoparticles into 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 that are 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 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 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.

Prevention of progression of age-related macular degeneration through photoprotection

Meyers, Ostrovsky, Kisseleva, Bonner; in collaboration with Avetisov, Chew, de Monasterio

The associations of age-related macular degeneration (AMD) with cataracts, earlier cataract surgery, cumulative exposure to sunlight, and pigmentation support the hypothesis that chronic photochemical injury plays a role in macular changes with age and AMD progression. Highly fluorescent lipofuscin granules accumulate with age in the retinal pigment epithelium (RPE) and co-localize with acute photosensitization of reactive oxygen intermediates (ROI) in the primate retina. The granules contain at least 10 fluorescent photochemical products, including 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 receptor outer segments (ROS) in normal daylight (rhodopsin bleaching). Although RPE lysosomal processing digests over 99 percent of the shed ROS contents, A2E and related fluorophores are not digested but rather are concentrated into lipofuscin granules (secondary lysosomes). By age 60, the average concentration of A2E within RPE cells reaches about 400 μmolar in normal eyes, well above levels that are toxic to cellular membranes. We hypothesize that both the segregation of A2E into lipofuscin granules and prevention of its redistribution into critical membranes are required for RPE health and believe that blue light-induced photo-oxidation of A2E within these granules is a critical mechanism of A2E cytotoxicity.

In collaboration with NEI and other laboratories, we developed a biophysical model using normal age-dependent 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. In our model, a decline of about one-third in the photo-oxidation action spectra-weighted macular irradiance with each decade of life, coupled with a nearly constant production rate of A2E-related fluorophores in the RPE, results in a nearly linear age-related increase in A2E concentrations in the RPE. A similar age-dependence of total lipofuscin granule volume and total fluorescence per RPE cell has been reported in human cadaver eyes. Total 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 complex. However, if the RPE/BM complex were the site of photo-oxidative injury driving AMD progression, the magnitude and rate of such injury would be expected to increase dramatically following cataract removal and intraocular lens (IOL) implantation, for which there is little clinical evidence.

Consequently, we propose a novel hypothesis that photochemically induced singlet oxygen generation within RPE lipofuscin granules induces 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 A2E to form A2E epoxides, which 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 model of macular aging reproduces the normal age dependence of lipofuscin and A2E and suggests that, 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 increased 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 and allows [A2E]_ss to rise. If our model is correct, then restoring or optimizing the spectral balance with the use of spectrally selective sunglasses could significantly lower A2E levels and may prevent associated macular degeneration.

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 photo-protective filters. We designed vermilion sunglasses that should optimally reduce both rod activation in bright ambient light and 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 the progression of both early and moderate AMD following cataract surgery and IOL implantation and in young patients with Stargardt's macular dystrophy.

¹ Russian Academy of Sciences, Moscow, Russia

COLLABORATORS

Sergei Avetisov, MD, DSc, Institute of Eye Diseases of the Russian Academy of Medical Sciences, Moscow, Russia
Emily Chew, MD, 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
Rose G. Mage, PhD, Laboratory of Immunology, NIAID, Bethesda, MD
Sanford Meyers, MD, Retina Consultants, Des Plaines, IL
Tom Pohida, MSEE, Computational Biology and Electronics Laboratory, CIT, Bethesda, MD
Dan Sackett, PhD, Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, MD

For further information, contact bonnerr@mail.nih.gov.