NOTE: New end date is 9/30/2010 per C. Guidry/JSC (6/2010)

In order to assess the time course of the expression and the natural history of the developing lesion, two experimental models of the lens are proposed in this application. Differentiating human lens cells in vitro will be used to extend the investigation of molecular mechanisms of action that follow particle radiation induced changes in key marker proteins using beams defined by NASA's operational parameters, e.g. low doses and dose-rates of protons or heavy ions. Mice will be irradiated and early lenticular changes associated with molecular aggregation will be followed using an exquisitely sensitive dynamic light scattering method to map the unique signatures of particle radiation-induced lens opacification. Information gathered from these measurements will be used to determine when the lenses will be harvested for protein identification. The results of the proposed research will improve mechanistic understanding underlying variability of dose-rate dependencies for cataract formation, and reduce the uncertainties in cataract risk assessment.

We examined the expression profile of a similar set of extraceullar matrix and adhesion molecules in whole lenses from Sprague-Dawley rats collected nine months after exposure to 10 or 100 cGy of 600 MeV/u iron ions. The results were compared to a set of control sham-treated lenses that were confirmed to be non-cataractous. Detailed visual inspection of iron-ion irradiated lenses at harvest confirms that the cataractous lesions are multifocal and polymorphic, with anterior cortical opacification, and anterior cortical involvements. Opacities appear to aggregate in the equatorial periphery. In addition, posterior subcapsular cataractous (PSC) dots are clearly visible in 50% of the lenses that were examined ex vivo. Results from focused microarray analysis revealed that lenses from 10 cGy-iron-ion-irradiated rat lenses showed significant 3- to 4-fold increases in genes in CAM & ECM functional groups in lenses. On the other hand, 100 cGy-iron-ion-irradiated rat lenses show a completely different gene response. Only 2 genes in the ECM & CAM series were increased 2- to 3-fold in the high-dose irradiated cataractous lenses, and two CAM series genes were significantly down-regulated 2- to 3-fold nine months after exposure. The high responsiveness of ECM gene families after a single low radiation dose to the lens in vivo contrasts with the decreased gene expression seen after a 10-fold higher radiation dose. The fact that such changes were persistent nine months after the exposures is unexpected. Clearly, different genes are affected at each dose level. It is not yet known how this gene profile correlates with the status of the opacifications observed at each dose at the same time post-exposure.

CD44, a cell-surface glycoprotein and a member of the cell adhesion molecule was shown to be a candidate that was upregulated at 8h after 100 cGy protons. Misregulation of this gene was also found in rat lenses >9 months after 10 cGy iron ions. CD44 in rat lenses have previously been reported and surgical removal of lens fiber cells from adult mice often results in a robust upregulation of CD44 protein, which preceded the upregulation of alpha-smooth muscle actin expression typically used as a marker of epithelial-mesenchyma transition in this model of lens epithelial cell fibrosis. CD44 protein has also been shown to increase in the lens epithelium after injury and is rodent-strain dependent. The CD44 family of receptors includes multiple variant isoforms due to post-translational modification and alternative splicing. Several isoforms have been linked to malignant properties, including migration, invasion and metastasis. Our Western results with isoform specific antibodies to probe for translation of CD44 variant gene product suggest that although CD44 protein levels were reduced 1 h after a small 10 cGy of protons, the v6 variant levels remained similar to the control levels. On the other hand, while the CD44 levels were elevated at 1.5 h after 100 cGy Titanium, the v6 variant also remained similar to the control levels. C57Bl6 male mice were sham-treated or exposed to 10, 50, 100 and 200 cGy of 600 MeV/u iron or 1 GeV/u protons in two separate large scale studies during NSRL08C and 09C. The animals were maintained for a period of 12 – 18 months post irradiation to measure (1) body weight changes, (2) changes in lens clarity using (a) slit lamp, and (b) quasi-elastic light scattering (QLS) technique. Lens tissues were also ex vivo imaged using stereo microscopy to document lens pathology and preserved for molecular analysis. Our collaborator Dr. Lee E. Goldstein has developed an analytical method to deconvolute the non-invasive infrared quasi-elastic light scattering and intensity fluctuation measurements to generate an autocorrelation function tau. The time constant for tau is an indicator of the size of the protein aggregation and light scatting centers in the lens. To date, comparisons have been made to show if the early in vitro human lens cell responses within hours after exposure correlate with the persistent in vivo whole rat lens response profile nine months after low doses. A comprehensive investigation of the dose-dependent expression of ECM and cell adhesion molecules (CAM) genes in rat, murine and human lens cells is ongoing for final preparation of a manuscript comparing the responses in three species.

NOTE: New end date is 9/30/2010 per C. Guidry/JSC (6/2010)

In order to assess the time course of the expression and the natural history of the developing lesion, two experimental models of the lens are proposed in this application. Differentiating human lens cells in vitro will be used to extend the investigation of molecular mechanisms of action that follow particle radiation induced changes in key marker proteins using beams defined by NASA's operational parameters, eg. low doses and dose-rates of protons or heavy ions. Mice will be irradiated and early lenticular changes associated with molecular aggregation will be followed using an exquisitely sensitive dynamic light scattering method to map the unique signatures of particle radiation-induced lens opacification. Information gathered from these measurements will be used to determine when the lenses will be harvested for protein identification. The results of the proposed research will improve mechanistic understanding underlying variability of dose-rate dependencies for cataract formation, and reduce the uncertainties in cataract risk assessment.

We have made significant progress in the completion of Specific Aims since our last progress report.

In vivo studies

Baseline measurements using a novel and proprietary quasi-elastic light scattering (QLS) technique and slit lamp examinations on lens clarity was made in animals prior to a low or a high dose of protons or iron ions at NSRL. After exposure, animals are monitored at regular intervals for body weight changes. Fully dilated animal lenses were evaluated monthly using QLS and slit lamp techniques.

To date, we have accumulated 9 months of QLS and slit lamp data from all animals. We have detected corneal abrasions randomly distributed in all study groups. Our collaborator Dr. Lee E. Goldstein has developed an analytical method to deconvolute the non-invasive infrared quasi-elastic light scattering and intensity fluctuation measurements to generate an autocorrelation function tau. The time constant for tau is an indicator of the size of the protein aggregation and light scatting centers in the lens. A computer program has been written to automate the autocorrelation analyses. Preliminary autocorrelation data analysis by Dr. Goldstein’s group show very promising early results demonstrating detectable changes in light scattering parameters in lenses that are determined to be clear through the conventional slit lamp techniques. The study is on-going and we expect that the results will be available for tabulation and analysis within the next 6 months. Our current plans are to harvest lenses from all the animals when we observe frank opacities in the high dose groups. Lenses will be ex vivo imaged using the stereo microscopic technique to document lens pathology. Some of the lenses will be frozen for molecular analysis while others will be fixed for immunohistochemistry.

In vitro studies

HLE cells were irradiated with a range of single doses of 1 GeV/u protons or 1 GeV/u titanium ions. Total RNA, protein and immunoflourescent samples were harvested from sham-treated controls and irradiated samples as a function of time post irradiation. Using a high throughput quantitative RT-PCR approach, we profiled the expression of 84 human genes that are known to be important for cell-cell and cell-matrix interactions. Genes in this panel include extracellular matrix (ECM) proteins associated with basement membrane constituents, collagens, and genes playing a role in ECM structure. Proteases involved in remodeling of the ECM are included as well as their inhibitors. This array also represents molecules important to cell adhesion, including molecules involved in cell-cell and cell-matrix adhesion, transmembrane molecules, and others.

Our data indicate that there are both qualitative and quantitative changes in the regulation of extracellular matrix genes after a low or a high dose of protons. Such transcriptional changes are also temporally regulated within a time-frame of up to 8 hrs post irradiation. We have also demonstrated that the quality of radiation plays a role in the transcriptional regulation of expression of ECM-associated genes with a significant number of down-regulated genes after exposure to either 10 cGy or 100 cGy Titanium ion.

In order to assess the time course of the expression and the natural history of the developing lesion, two experimental models of the lens are proposed in this application. Differentiating human lens cells in vitro will be used to extend the investigation of molecular mechanisms of action that follow particle radiation induced changes in key marker proteins using beams defined by NASA's operational parameters, eg. low doses and dose-rates of protons or heavy ions. Mice will be irradiated and early lenticular changes associated with molecular aggregation will be followed using an exquisitely sensitive dynamic light scattering method to map the unique signatures of particle radiation-induced lens opacification. Information gathered from these measurements will be used to determine when the lenses will be harvested for protein identification. The results of the proposed research will improve mechanistic understanding underlying variability of dose-rate dependencies for cataract formation, and reduce the uncertainties in cataract risk assessment.

We have recently acquired quantitative PCR (Q-PCR gene expression array data in whole lenses from Sprague-Dawley male and female rats (6-12 weeks of age at time of exposure) irradiated with 600 MeV/u iron ions in our earlier funding period. These lenses were collected nine months after radiation exposure. mRNA isolated from the whole irradiated lenses was analyzed for quantitative changes in gene expression with a Rat Extracellular Matrix and Adhesion Molecules RT2 Profiler Array (SABiosciences, MD). Fold-changes in gene expression were considered significant at the p < 0.09 level. Quantitative analysis of 96 genes included in the array were conducted on lens samples obtained from animals exposed to 10 cGy or 100 cGy of 600 MeV/amu iron-ions. The results were normalized to the unirradiated sham-treated control lenses. Each analysis included mRNA of lenses from 4 animals harvested nine months after the irradiation.

Detailed visual inspection of iron-ion irradiated lenses confirms that the cataractous lesions are multifocal and polymorphic, with anterior cortical opacification, and anterior cortical involvements. Opacities appear to aggregate in the equatorial periphery suggesting likely transition at the lens bow region. In addition, posterior subcapsular catactous (PSC) dots are clearly visible in 50% of the lenses that were examined ex vivo.

Analyses of individual rat lenses for quantitative gene expression show remarkable reproducibility within control and treatment groups. Results from array analysis revealed that lenses from 10 cGy-iron-ion-irradiated rat lenses showed significant 3- to 4-fold increases in genes in CAM (Cell Adhesion Molecules) & ECM (Extra Cellular Matrix) functional groups in lenses with early cataractous changes nine months after exposure, including > 3-fold changes in the CD44 antigen, a gene that was previously noted in epithelial cells in cataractous human lenses (Nishi et al, IOVS, 1997). On the other hand, 100 cGy-iron-ion-irradiated rat lenses show a completely different gene response. Only 2 genes in the ECM & CAM series were increased 2- to 3-fold in cataractous lenses, and two CAM series genes were significantly down-regulated 2- to 3-fold nine months after exposure.

The high responsiveness of ECM gene families demonstrated after a low radiation dose to the lens in vivo contrasts with the decreased gene expression seen after a 10-fold higher radiation dose nine months after the exposures. Clearly, different genes are affected at each dose level. It is not yet known how this gene profile correlates with the status of the opacifications observed at each dose at the same time post-exposure.

We participated in two separate NSRL runs during the first year of this grant. In NSRL08A, HLE cells were grown on matrix-coated plastic tissue culture flasks and irradiated with 1 GeV/u protons or 1 GeV/u titanium. Single doses of 10, 50 or 100 cGy were given. Total RNA and protein extracts from sham-treated control and irradiated samples were harvested at different times after radiation exposure and processed. Samples were also fixed with 4 % paraformaldehyde and stored until analysis. Another cell study was also conducted during NSRL 08C to duplicate the proton experiment performed during the 08A campaign. C57Bl6 male mice were whole-body irradiated during NSRL 08C. Animals were purchased from Charles River Laboratories, shipped to Dr. Lee Goldstein’s laboratory at Boston University for baseline quasi-elastic light scattering (QLS) evaluation prior to transporting to the animal facility at BNL. Twenty five animals in each dose/ion group were whole-body exposed with a single 10 or 100 cGy of 1 GeV/u protons or 1 GeV/u Titanium ions. Within a week after exposure, animals were shipped to the animal facility at LBNL for long-term maintenance and evaluations. Planned monthly QLS evaluations and slit lamp video monitoring are in progress. We aim to follow these animals up to 1 year after the radiation exposure. Our current plans are to harvest lenses from all the animals when we observe frank opacities in the high dose groups. Lenses will be ex vivo photographed to document lens pathology. Some of the lenses will be frozen for molecular analysis while others will be fixed for immunohistochemistry.

Work is in progress to show if the early in vitro human lens cell responses within hours after exposure correlate with the persistent in vivo whole rat lens response profile nine months after low doses. Integrin alpha 5 shows good cross-species induction after 1 Gy doses of 600 Mev/amu Iron to rats, and 4 Gy doses to cultured differentiating human lens cells. A comprehensive investigation of the dose-dependent expression of ECM and CAM genes in rat and human lens cells is ongoing. Studies using lower particle fluences are planned.

In order to assess the time course of the expression and the natural history of the developing lesion, two experimental models of the lens are proposed in this application. Differentiating human lens cells in vitro will be used to extend the investigation of molecular mechanisms of action that follow particle radiation induced changes in key marker proteins using beams defined by NASA's operational parameters, eg. low doses and dose-rates of protons or heavy ions. Mice will be irradiated and early lenticular changes associated with molecular aggregation will be followed using an exquisitely sensitive dynamic light scattering method to map the unique signatures of particle radiation-induced lens opacification. Information gathered from these measurements will be used to determine when the lenses will be harvested for protein identification. The results of the proposed research will improve mechanistic understanding underlying variability of dose-rate dependencies for cataract formation, and reduce the uncertainties in cataract risk assessment.