Determining RBE for development of lung fibrosis induced by fractionated irradiation with carbon ions utilizing fibrosis index and high-LET BED model.

BACKGROUND AND PURPOSES: Carbon ion radiotherapy (CIRT) with raster scanning technology is a promising treatment for lung cancer and thoracic malignancies. Determining normal tissue tolerance of organs at risk is of utmost importance for the success of CIRT. Here we report the relative biological effectiveness (RBE) of CIRT as a function of dose and fractionation for development of pulmonary fibrosis using well established fibrosis index (FI) model. MATERIALS AND METHODS: Dose series of fractionated clinical quality CIRT versus conventional photon irradiation to the whole thorax were compared in C57BL6 mice. Quantitative assessment of pulmonary fibrosis was performed by applying the FI to computed tomography (CT) data acquired 24-weeks post irradiation. RBE was calculated as the ratio of photon to CIRT dose required for the same level of FI. Further RBE predictions were performed using the derived equation from high-linear energy transfer biologically effective dose (high-LET BED) model. RESULTS: The averaged lung fibrosis RBE of 5-fraction CIRT schedule was determined as 2.75 ±â€¯0.55. The RBE estimate at the half maximum effective dose (RBEED50) was estimated at 2.82 for clinically relevant fractional sizes of 1-6 Gy. At the same dose range, an RBE value of 2.81 ±â€¯0.40 was predicted by the high-LET BED model. The converted biologically effective dose (BED) of CIRT for induction of half maximum FI (BEDED50) was identified to be 58.12 Gy3.95. In accordance, an estimated RBE of 2.88 was obtained at the BEDED50 level. The LQ model radiosensitivity parameters for 5-fraction was obtained as αH = 0.3030 ±â€¯0.0037 Gy-1 and ßH = 0.0056 ±â€¯0.0007 Gy-2. CONCLUSION: This is the first report of RBE estimation for CIRT with the endpoint of pulmonary fibrosis in-vivo. We proposed in present study a novel way to mathematically modeling RBE by integrating RBEmax and α/ßL based on conventional high-LET BED conception. This model well predicted RBE in the clinically relevant dose range but is sensitive to the uncertainties of α/ß estimates from the reference photon irradiation (α/ßL). These findings will assist to eliminate current uncertainties in prediction of CIRT induced normal tissue complications and builds a solid foundation for development of more accurate in-vivo data driven RBE estimates.

Modeling and multiscale characterization of the quantitative imaging based fibrosis index reveals pathophysiological, transcriptome and proteomic correlates of lung fibrosis induced by fractionated irradiation.

Pulmonary fibrosis represents a leading cause of morbidity and mortality worldwide. Therapy induced lung fibrosis constitutes a pivotal dose-limiting side effect of radiotherapy and other anticancer agents. We aimed to develop objective criteria for assessment of fibrosis and discover pathophysiological and molecular correlates of lung fibrosis as a function of fractionated whole thoracic irradiation. Dose-response series of fractionated irradiation was utilized to develop a non-invasive and quantitative measure for the degree of fibrosis - the fibrosis index (FI). The correlation of FI with histopathology, blood-gas, transcriptome and proteome responses of the lung tissue was analyzed. Macrophages infiltration and polarization was assessed by immunohistochemistry. Fibrosis development followed a slow kinetic with maximum lung fibrosis levels detected at 24-week post radiation insult. FI favorably correlated with radiation dose and surrogates of lung fibrosis i.e., enhanced pro-inflammatory response, tissue remodeling and extracellular matrix deposition. The loss of lung architecture correlated with decreased epithelial marker, loss of microvascular integrity with decreased endothelial and elevated mesenchymal markers. Lung fibrosis was further attributed to a switch of the inflammatory state toward a macrophage/T-helper cell type 2-like (M2/Th2) polarized phenotype. Together, the multiscale characterization of FI in radiation-induced lung fibrosis (RILF) model identified pathophysiological, transcriptional and proteomic correlates of fibrosis. Pathological immune response and endothelial/epithelial to mesenchymal transition were discovered as critical events governing lung tissue remodeling. FI will be instrumental for deciphering the molecular mechanisms governing lung fibrosis and discovery of novel targets for treatment of this devastating disease with an unmet medical need.

Quantitative assessment of radiation dose and fractionation effects on normal tissue by utilizing a novel lung fibrosis index model.

BACKGROUND: Normal lung tissue tolerance constitutes a limiting factor in delivering the required dose of radiotherapy to cure thoracic and chest wall malignancies. Radiation-induced lung fibrosis (RILF) is considered a critical determinant for late normal tissue complications. While RILF mouse models are frequently approached e.g., as a single high dose thoracic irradiation to investigate lung fibrosis and candidate modulators, a systematic radiobiological characterization of RILF mouse model is urgently needed to compare relative biological effectiveness (RBE) of particle irradiation with protons, helium-, carbon and oxygen ions now available at HIT. We aimed to study the dose-response relationship and fractionation effect of photon irradiation in development of pulmonary fibrosis in C57BL/6 mouse. METHODS: Lung fibrosis was evaluated 24 weeks after single and fractionated whole thoracic irradiation by quantitative assessment of lung alterations using CT. The fibrosis index (FI) was determined based on 3D-segmentation of the lungs considering the two key fibrosis parameters affected by ionizing radiation i.e., a dose/fractionation dependent reduction of the total lung volume and increase of the mean lung density. RESULTS: The effective dose required to induce 50% of the maximal possible fibrosis (ED 50 ) was 14.55 ± 0.34Gy and 27.7 ± 1.22Gy, for single and five- fractions irradiation, respectively. Applying a deterministic model an α/ß = 4.49 ± 0.38 Gy for the late lung radiosensitivity was determined. Intriguingly, we found that a linear-quadratic model could be applied to in-vivo log transformed fibrosis (FI) vs. irradiation doses. The LQ model revealed an α/ß for lung radiosensitivity of 4.4879 Gy for single fraction and 3.9474 for 5-fractions. Our FI based data were in good agreement with a meta-analysis of previous lung radiosensitivity data derived from different clinical endpoints and various mouse strains. The effect of fractionation on RILF development was further estimated by the biologically effective dose (BED) model with threshold BED (BED Tr ) = 30.33 Gy and BED ED50  = 61.63 Gy, respectively. CONCLUSION: The systematic radiobiological characterization of RILF in the C57BL/6 mouse reported in this study marks an important step towards precise estimation of dose-response for development of lung fibrosis. These radiobiological parameters combined with a large repertoire of genetically engineered C57BL/6 mouse models, build a solid foundation for further biologically individualized risk assessment of RILF and functional RBE prediction on novel of particle qualities.

Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat.

Liver regeneration requires spatially and temporally precisely coordinated proliferation of the two major hepatic cell populations, hepatocytes and liver sinusoidal endothelial cells (LSECs), to reconstitute liver structure and function. The underlying mechanisms of this complex molecular cross-talk remain elusive. Here, we show that the expression of Angiopoietin-2 (Ang2) in LSECs is dynamically regulated after partial hepatectomy. During the early inductive phase of liver regeneration, Ang2 down-regulation leads to reduced LSEC transforming growth factor-ß1 production, enabling hepatocyte proliferation by releasing an angiocrine proliferative brake. During the later angiogenic phase of liver regeneration, recovery of endothelial Ang2 expression enables regenerative angiogenesis by controlling LSEC vascular endothelial growth factor receptor 2 expression. The data establish LSECs as a dynamic rheostat of liver regeneration, spatiotemporally orchestrating hepatocyte and LSEC proliferation through angiocrine- and autocrine-acting Ang2, respectively.

RANGE: Gene Transfer of Reversibly Controlled Polycistronic Genes.

We developed a single vector recombinant adeno-associated viral (rAAV) expression system for spatial and reversible control of polycistronic gene expression. Our approach (i) integrates the advantages of the tetracycline (Tet)-controlled transcriptional silencer tTS(Kid) and the self-cleaving 2A peptide bridge, (ii) combines essential regulatory components as an autoregulatory loop, (iii) simplifies the gene delivery scheme, and (iv) regulates multiple genes in a synchronized manner. Controlled by an upstream Tet-responsive element (TRE), both the ubiquitous chicken ß-actin promoter (CAG) and the neuron-specific synapsin-1 promoter (Syn) could regulate expression of tTS(Kid) together with two 2A-linked reporter genes. Transduction in vitro exhibited maximally 50-fold regulation by doxycycline (Dox). Determined by gene delivery method as well as promoter, highly specific tissues were transduced in vivo. Bioluminescence imaging (BLI) visualized reversible "ON/OFF" gene switches over repeated "Doxy-Cycling" in living mice. Thus, the reversible rAAV-mediated N-cistronic gene expression system, termed RANGE, may serve as a versatile tool to achieve reversible polycistronic gene regulation for the study of gene function as well as gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e85; doi:10.1038/mtna.2013.15; published online 9 April 2013.

Osteoporosis influences osteogenic but not angiogenic response during bone defect healing in a rat model.

Angiogenesis is pivotal for bone metabolism and bone defect healing. Yet the role of vascularization in osteoporosis and osteoporotic bone repair mechanisms is unclear. Here we investigated effects of osteoporotic phenotype on vascularization during bone defect healing in a rodent osteotomy model using volumetric computed tomography (VCT), dynamic contrast-enhanced VCT (DCE-VCT), dynamic contrast-enhanced MRI (DCE-MRI) and histology. In 16 rats, 8 with physiological bone status (SHAM) and 8 with osteoporotic bone status induced by ovariectomy (OVX) in combination with a vitamin D- and low calcium diet, wedge-shaped defects were created at the left distal femur and stabilized internally by T-shaped miniplate. MRI and VCT were performed in all animals 6 weeks after this procedure. By VCT, relative bone density in the defect was evaluated. Using DCE-VCT and DCE-MRI, parameters associated with regional blood volume were calculated in the bone defect, vicinity of the defect, surrounding muscles and bone marrow: Amplitude A and exchange rate constant Kep (DCE-MRI, respectively) as well as peak enhancement PE and area under the curve AUC (DCE-VCT, respectively). In animals of osteoporotic phenotype, bone density within the osseous defect was significantly reduced as compared to SHAM rats. Vascularization parameters determined by DCE-MRI and DCE-VCT in the defect were significantly elevated compared to the adjacent tissues for both SHAM and OVX groups. However, comparing SHAM and OVX rats, no statistically different values were found by DCE-MRI and DCE-VCT concerning any determined vascularization parameter within the bone defect. Furthermore, parameters of vascularization were increased for OVX as compared to SHAM rats within the bone marrow although significant difference was only found for A. In a rat osteotomy model we showed that at the reparative healing stage, osteoporotic phenotype did influence osteogenic but not angiogenic response within bone defect as imaged by DCE-MRI and DCE-VCT. This study provides insight into the relationship between angiogenesis and osteogenesis during osteoporosis-related compromised bone healing.

Quantitative assessment of microcirculation and diffusion in the bone marrow of osteoporotic rats using VCT, DCE-MRI, DW-MRI, and histology.

BACKGROUND: Etiologic and pathophysiologic role of functional bone marrow processes is not fully understood especially in the case of osteoporosis. PURPOSE: To investigate the role of vascularization and diffusion in rat models of osteoporosis through a cross-correlation between non-invasive in-vivo imaging and invasive ex-vivo imaging of bone, bone marrow, and in particular of microcirculation. MATERIAL AND METHODS: Osteoporosis was induced in rats by combining ovariectomy (OVX) with calcium and Vitamin D3 deficiency, or with glucocorticoid (dexamethasone). For comparison, controls underwent a sham surgery. In in-vivo investigations, animals (n = 36) were examined by volumetric CT (VCT) and MRI at 1, 3, or 12 months post surgery. Using VCT, bone morphology was monitored and relative bone density r within pelvis was extracted. With DCE-MRI and DW-MRI, parameters A (amplitude), Kep (exchange rate constant), and ADC (apparent diffusion coefficient) were acquired for regions of lumbar vertebrae, pelvis, and femur. In ex-vivo investigations, selective histological sections of pelvis were either stained with hematoxylin and eosin (HE stain) for quantifying vessel size and density or immunostained for collagen IV and α-smooth muscle actin to assess vessel maturity (SMA/collagen IV ratio). RESULTS: After 12 months, decrease in DCE-MRI parameter Kep was found in all locations of osteoporotic rats (strongest in femur and lumbar vertebrae) while no significant differences were seen for parameter A and DW-MRI parameter ADC. Furthermore, vessel rarefication and maturation were observed on the histological level in animals with osteoporotic phenotype. In particular in the pelvis, the osteoporotic individuals (irrespective of the osteoporosis inducers applied) exhibited decreased Kep, significantly reduced vessel density, significantly increased vessel maturity, as well as statistically unaltered A, ADC, and vessel diameter. CONCLUSION: Changes in microcirculation but not diffusion in the bone marrow of osteoporotic rats are detected by DCE-MRI and DW-MRI due to vessel rarefication and maturation.

Calibration of cone beam CT using relative attenuation ratio for quantitative assessment of bone density: a small animal study.

PURPOSE: Cone beam computed tomography (CBCT) has the disadvantage of providing non-quantitative results for bone density determination. The aim of this study is to calibrate CBCT results by using an internal reference (such as muscle) for quantitatively assessing bone density. METHODS: We developed a new method using the relative attenuation ratio between two nearby materials (such as bone and muscle) for systemic error correction in CBCT that depends on the relative object position in the image volume. Phantom calibration was performed to calculate the acquired attenuation ratio in Hounsfield units (HU), comparable to the results from clinical multislice spiral computed tomography (MSCT). In addition, a small animal study with an osteoporotic rat model was evaluated to show the feasibility of this presented method to quantitatively assess bone density using a CBCT system. RESULTS: The phantom study results showed that the calibration process successfully corrected the systemic inaccuracy from CBCT, and the calibrated HU values agreed with the values measured from MSCT. In the small animal study, the quantitative bone densities assessed from the calibrated CBCT results were consistent with the results from MSCT data. CONCLUSION: A practical method to quantitatively estimate attenuation (HU) values for bone tissues from CBCT scans that are comparable to MSCT scans is proposed. The method may improve the quantification ability of CBCT scanning without any additional hardware requirements.

Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy.

Innovative and personalized therapeutic approaches result from the identification and control of individual aberrantly expressed genes at the transcriptional and post-transcriptional level. Therefore, it is of high interest to establish diagnostic, therapeutic and theranostic strategies at these levels. In the present study, we used the Diels-Alder Reaction with inverse electron demand (DAR(inv)) click chemistry to prepare a series of cyclic RGD-BioShuttle constructs. These constructs carry the near-infrared (NIR) imaging agent Cy7 and the chemotherapeutic agent temozolomide (TMZ). We evaluated their uptake by and their efficacy against integrin α(v)ß(3)-expressing MCF7 human breast carcinoma cells. In addition, using a mouse phantom, we analyzed the suitability of this targeted theranostic agent for NIR optical imaging. We observed that the cyclic RGD-based carriers containing TMZ and/or Cy7 were effectively taken up by α(v)ß(3)-expressing cells, that they were more effective than free TMZ in inducing cell death, and that they could be quantitatively visualized using NIR fluorescence imaging. Therefore, these targeted theranostic agents are considered to be highly suitable systems for improving disease diagnosis and therapy.

Digital eversion of a hollow structure: an application in virtual colonography.

A new methodology is presented for digital eversion of a hollow structure. The digital eversion is advantageous for better visualization of a larger portion of the inner surface with preservation of geometric relationship and without time-consuming navigation. Together with other techniques, digital eversion may help improve screening, diagnosis, surgical planning, and medical education. Two eversion algorithms are proposed and evaluated in numerical simulation to demonstrate the feasibility of the approach.