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Glycogen Synthase Kinase 3β Participates in Function of Vascular Endothelium
S472
I. J. Radiation Oncology
● Biology ● Physics
Volume 63, Number 2, Supplement, 2005
profound alterations in the development of kidney dysfunction. Surprisingly, EPO increased the loss of kidney function, whereas IGF-1 acted as a protective agent after high-dose irradiation. We have examined the IGF-1 dose-response and defined the best dose per injection. Established renal insufficiency did not improve after prolonged administration of IGF-1, suggesting that early intervention might be the preferable approach. The next step will be to determine the dose-modifying factor for IGF-1 treatment.
2408
Antibody to avb6 Integrin Prevents Radiation-Induced Lung Fibrosis
S.C. Formenti,1 S. Jacoby,2 K. Grant,2 G. Horan,2 . Weinreb,2 M. Devitt,1 J. Munger2 Radiation Oncology, New York University, New York, NY, 2Medicine, New York University, NY, NY
1
Purpose/Objective: The development of pulmonary fibrosis limits the dose of therapeutic lung radiation. The pleiotrophic cytokine TGFb is thought to be central to this process and to other fibrotic diseases. The integrin avb6 activates TGFb from its latent form, and plays an important role in localizing TGFb effects; it is dramatically upregulated on lung epithelial cells before fibrosis develops after injury. Furthermore, knockout mice that lack avb6 are protected from lung fibrosis after radiation exposure, as well as from other fibrotic stimuli such as bleomycin. This makes inhibition of the avb6 integrin a possible therapeutic target and, to that end, a monoclonal antibody has been developed. Blockade at another level in this pathway by soluble TGFb receptor, which inhibits all TGFb activity, prevents early radiation pneumonitis and bleomycin-induced fibrosis. We compared the effects of anti-avb6 mAb and soluble TGFb receptor on the late pulmonary fibrotic response after thoracic radiation in a mouse model. Materials/Methods: We injected 149 C57BL/6 mice with various doses of anti-avb6 antibodies (0.3 mg/kg, 1 mg/kg and 10mg/kg), soluble TGFb receptor (5 mg/kg), or appropriate controls (PBS and IgG), starting 15 weeks after 14Gy thoracic radiation. Mice were monitored daily and sacrificed if in respiratory distress or moribund. We assessed fibrosis in mice sacrificed at 26 weeks by calculating the percent fibrosis area of paraffin-embedded, formalin-fixed tissue sections that were stained with Masson trichrome. Expression of avb6 was assessed by immunohistochemistry. Results: We found that the antibody caused a dose-dependent attenuation of fibrosis. Soluble TGFb receptor also inhibited fibrosis, but did not block the upregulation of avb6. Kaplan-Meier analysis showed no survival benefit as a result of avb6 blockade; in fact, there was a trend to excess mortality in the high-dose group (p⫽0.08). Conclusions: The prevention of pulmonary fibrosis by the late administration of either soluble TGFb receptor or antibody to avb6 clearly demonstrate that radiation-induced pulmonary fibrosis is a TGFb-dependent process. Although TGFb upregulates the avb6 integrin in vitro, we did not observe that blockade of TGFb activity altered avb6 expression in vivo. The 1 mg/kg dose of anti-avb6 but not the 0.3 mg/kg dose blocked fibrosis. However the trend to higher mortality with treatment with the higher dose antibody (10 mg/kg), if confirmed in further experiments, may indicate that partial rather than high-level inhibition of avb6-mediated TGFb activation may yield optimal results. These results support further investigation into blockade of avb6 as a therapeutic modality.
2409
Glycogen Synthase Kinase 3 Participates in Function of Vascular Endothelium
K.C. Cuneo, E. Yazlovitskaya, D. Thotala, D.E. Hallahan Radiation Oncology, Vanderbilt University, Nashville, TN Purpose/Objective: Akt signaling participates in angiogenesis in response to VEGF stimulation of endothelial cells and regulates capillary-like tubule formation which requires the inactivation of GSK3b. Over-expression of GSK-3 induces apoptosis in various cells in culture, and specific inhibitors of GSK-3 ameliorate this apoptotic process. We therefore studied the role of Akt/GSK3 signaling in irradiated vascular endothelium. Materials/Methods: Radiation-induced phosphorylation of Akt and GSK3 was studied in endothelial cells irradiated with 3 Gy. Total protein was extracted and Western immunoblots were probed with antibodies specific for phosphorylated Akt and total Akt. To determine whether Akt participates in GSK3 phosphorylation following irradiation, the dominant negative Akt genetic construct (Ad.⌬ Akt) was overexpressed in primary culture vascular endothelial cells. Western blot analysis of caspase 3 activation was utilized to verify the induction of apoptosis. Capillary tubule formation in matrigel was studied in irradiated vascular endothelium overexpressing Ad.⌬ Akt. Results: Phosphorylation of Akt was minimal in untreated control endothelial cells, but increased within 5 minutes and was maximal at 15 min. Because GSK3b is one of the key effectors of Akt signaling, radiation may also induce phosphorylation of GSK3b. Similarly, minimal phosphorylation of GSK3b was found in untreated control cells, but increased at 5 minutes and was maximal at 15 min. The dominant negative Akt genetic construct (Ad.⌬ AKT) was overexpressed in endothelial cells. Overexpression of Ad.⌬ AKT abrogated GSK3 phosphorylation following irradiation. In comparison, cells transduced with control vector Ad.GFP showed no interruption of radiation-induced GSK3 phosphorylation. 5% of primary culture endothelial cells undergo apoptosis following treatment with Ad.⌬ AKT as compared to 2.5% in the control cells. In comparison, when irradiated, Ad.⌬ AKT transduced cells had a markedly increased percentage of apoptotic cells to 25%, which was significantly increased as compared to treatment with Ad.⌬ AKT or radiation alone (p⬍ 0.001). Caspase 3 cleavage was induced in HUVEC transduced with Ad.⌬ AKT followed by irradiation. In comparison, Ad.⌬ Akt or 3 Gy resulted in minimal caspase 3 cleavage in endothelial cells. We next examined caspase 3 cleavage and propidium iodide staining, which showed that GSK3b dominant negative prevented apoptosis. Dominant negative GSK3b transduced cells were then treated with Akt inhibitor and radiation, which decreased the caspase 3 activity in irradiated endothelial cells treated with Akt inhibitor. In comparison, cells treated with the Ad.GFP control vector showed increased caspase 3 cleavage following treatment with Akt inhibitor and radiation. To verify the role of GSK3b signaling during in vivo microvascular response, we studied capillary formation in the irradiated mouse utilizing VEGF within matrigel implanted subcutaneously into the mouse flank. The mouse flank was then irradiated with 3 Gy and FITC-dextran was injected by tail vein 72 hours later. The FITC-dextran containing capillaries within the Matrigel plug were imaged by fluorescence microscopy. Capillary formation within Matrigel occurred within the irradiated
Proceedings of the 47th Annual ASTRO Meeting
mouse flank. Ad.⌬ Akt and Ad.⌬ GSK3b were added to Matrigel and capillaries were counted by microscopy. The Akt dominant negative entirely eliminated capillary-like tubule formation. In comparison, the GSK3b dominant negative reversed the effect of Akt mutant on capillary formation (P⫽0.04). Conclusions: These data indicate that ionizing radiation-induced Akt signaling contributes to inactivation of GSK3b through phosphorylation of this kinase. GSK3 phosphorylation, in turn, impacts upon endothelial cell survival and capillary formation in mouse models.
2410
Gene Selection by a Genomic Radiation Sensitivity Classifier is Influenced by the Presence of a Mutated ras Gene
J.F. Torres-Roca, S. Eschrich, T. Yeatman Interdisciplinary Oncology, H Lee Moffitt Cancer Center, Tampa, FL Purpose/Objective: In previous studies, we demonstrated the development of a gene expression radiation sensitivity predictor in tumor cell lines, that accurately predicted the survival fraction at 2 Gy (SF2) in 22 of 35 cell lines from the NCI panel of 60. This was accomplished using baseline gene expression profiles derived from microarrays (Affymetrix HU-6800) that were available in the literature. In the classifier design, we treated radiation sensitivity as a continuous variable, and included a multivariate linear regression model as the predictive step. A leave-one-out cross validation model was used in the design. This analytical approach was validated biologically, by demonstrating that one of the selected predictive genes (RbAp48) was involved mechanistically in radiation response. In this work, we sought to determine the role that a mutated ras gene would convey on the baseline gene expression signature and how this would affect the process of radiation sensitivity prediction. Materials/Methods: A total of 48 cell lines from the NCI-panel of 60 were used for this study. SF2s were either obtained from the literature (23 cell lines) or determined in our laboratory using clonogenic assays (25 cell lines). Gene expression profiles were from Affymetrix HU-6800 (7,129 genes) from a previously published study. The gene expression data was preprocessed using the Affymetrix MAS 4.0 algorithm in average difference units. Negative expression values were set to zero and the chips were normalized to the same mean intensity. Ras status (mutated vs. wild-type) was obtained from the literature. Gene selection was done using SAM analysis. Radiation sensitivity (defined as SF2) was treated as a continuous variable. A leave-one-out cross-validation model was used in the classifier design. A multivariate linear regression model was used for radiosensitivity prediction. Results: We initially determined whether Ras status would convey a different baseline gene expression signature. Indeed, SAM analysis identified a subset of genes whose baseline expression separated cells lines by Ras status. Since baseline gene expression profiles were affected by the presence of a mutated Ras protein, we determined whether the genes selected by the radiation sensitivity classifier, were dependent or independent of Ras status. We stratified the original classifier by Ras status, and performed the gene selection step in each group of cell lines. Interestingly, the genes selected by each group were completely different. The three known genes (RbAp48, RSG-19, R5PIA) that had been selected by the overall classifier without Ras stratification and that were previously shown to be highly useful in predicting radiosensitivity, were highly ranked by the Ras mutated cell lines but not by the wild type lines, suggesting that the Ras mutated cell lines were driving the classification process. RbAp48, RSG-19 and R5PIA were ranked 19th, 46th and 262nd out of 7,129 genes by the Ras-mutated cell lines. In contrast wild-type cell lines ranked these genes 743rd, 758th and 397th respectively. When all cell lines were taken together, these three genes ranked in the top 10 genes (5th, 1st and 9th). Conclusions: The process of gene selection by a biologically-validated genomic radiation sensitivity classifier, is influenced by the presence of a mutated Ras protein. The translation of genomic predictive models to the clinic should take this observation into account.
2411
Non-Invasive Monitoring of Radiation Induced Liver Damage Using Spectroscopic Imaging
31
P Magnetic Resonance
C. Landis,2 L. Liu,1 D. Shafritz,2 J. Roy-Chowdhury,2 H. Hetherington,2 C. Guha1 1 Radiation Oncology, Montefiore Medical Center - Albert Einstein College of Medicine, Bronx, NY, 2Marion Bessin Liver Research Center, Bronx, NY Purpose/Objective: The combination of partial hepatectomy (PH) and whole liver irradiation (PHRT) leads to extensive radiation-induced liver damage (RILD) with 70 – 80% mortality in rats (1). Furthermore, hepatocyte transplantation ameliorated RILD in this model. We used 31P Magnetic Resonance Spectroscopic Imaging (MRSI) to non-invasively monitor changes in phosphorylated metabolites following PHRT as prognostic markers for RILD. Materials/Methods: F344 rats were subjected to 70% PH alone or PH followed by hepatic RT (50 Gy). For MR studies, a 9.4 T Varian INOVA MR system with a birdcage coil for 1H MRI and a surface coil for 31P MRS were used. MR acquisition was gated to the mechanical ventilation rate. A standard multi-slice spin echo imaging sequence was used for scout images (TE⫽25ms, TR⫽1s, FOV⫽64⫻64mm, ns⫽17). For 31P MRSI, a 45° nonselective excitation and 3D phase encoding using a spherical k-space sampling scheme (FOV⫽64⫻64x64mm, TR⫽1s). Data was processed using routines written in the MATLAB programming language. MRS studies were performed 1, 3, and 8 days after PH or PHRT (n⫽3– 4 rats/time point). Livers from sacrificed animals were analyzed for hepatocyte regeneration. Results: Compared to untreated controls (1.32 ⫾ 0.09), the ATP/Pi peak area ratios were significantly reduced in PH (0.87 ⫾ 0.11) and PHRT (0.70 ⫾ 0.04) groups at 1d. BrdU incorporation was markedly reduced in the PHRT group compared to PH alone. A representative MRI scout image and 31P MR spectra from 8 days after a) PH alone or b) PHRT is shown. The spectra are derived from MRSI data within the indicated region of interest. The peak assignments for various phosphorylated metabolites are also indicated. At 8 days, the PH only group exhibits a normalized ATP/Pi ratio (1.28 ⫾ 0.14), indistinguishable from untreated controls. In contrast, the ATP/Pi ratio did not return to baseline in the PHRT rats (1.03 ⫾ 0.07, p⬍0.03).
S473
I. J. Radiation Oncology
● Biology ● Physics
Volume 63, Number 2, Supplement, 2005
profound alterations in the development of kidney dysfunction. Surprisingly, EPO increased the loss of kidney function, whereas IGF-1 acted as a protective agent after high-dose irradiation. We have examined the IGF-1 dose-response and defined the best dose per injection. Established renal insufficiency did not improve after prolonged administration of IGF-1, suggesting that early intervention might be the preferable approach. The next step will be to determine the dose-modifying factor for IGF-1 treatment.
2408
Antibody to avb6 Integrin Prevents Radiation-Induced Lung Fibrosis
S.C. Formenti,1 S. Jacoby,2 K. Grant,2 G. Horan,2 . Weinreb,2 M. Devitt,1 J. Munger2 Radiation Oncology, New York University, New York, NY, 2Medicine, New York University, NY, NY
1
Purpose/Objective: The development of pulmonary fibrosis limits the dose of therapeutic lung radiation. The pleiotrophic cytokine TGFb is thought to be central to this process and to other fibrotic diseases. The integrin avb6 activates TGFb from its latent form, and plays an important role in localizing TGFb effects; it is dramatically upregulated on lung epithelial cells before fibrosis develops after injury. Furthermore, knockout mice that lack avb6 are protected from lung fibrosis after radiation exposure, as well as from other fibrotic stimuli such as bleomycin. This makes inhibition of the avb6 integrin a possible therapeutic target and, to that end, a monoclonal antibody has been developed. Blockade at another level in this pathway by soluble TGFb receptor, which inhibits all TGFb activity, prevents early radiation pneumonitis and bleomycin-induced fibrosis. We compared the effects of anti-avb6 mAb and soluble TGFb receptor on the late pulmonary fibrotic response after thoracic radiation in a mouse model. Materials/Methods: We injected 149 C57BL/6 mice with various doses of anti-avb6 antibodies (0.3 mg/kg, 1 mg/kg and 10mg/kg), soluble TGFb receptor (5 mg/kg), or appropriate controls (PBS and IgG), starting 15 weeks after 14Gy thoracic radiation. Mice were monitored daily and sacrificed if in respiratory distress or moribund. We assessed fibrosis in mice sacrificed at 26 weeks by calculating the percent fibrosis area of paraffin-embedded, formalin-fixed tissue sections that were stained with Masson trichrome. Expression of avb6 was assessed by immunohistochemistry. Results: We found that the antibody caused a dose-dependent attenuation of fibrosis. Soluble TGFb receptor also inhibited fibrosis, but did not block the upregulation of avb6. Kaplan-Meier analysis showed no survival benefit as a result of avb6 blockade; in fact, there was a trend to excess mortality in the high-dose group (p⫽0.08). Conclusions: The prevention of pulmonary fibrosis by the late administration of either soluble TGFb receptor or antibody to avb6 clearly demonstrate that radiation-induced pulmonary fibrosis is a TGFb-dependent process. Although TGFb upregulates the avb6 integrin in vitro, we did not observe that blockade of TGFb activity altered avb6 expression in vivo. The 1 mg/kg dose of anti-avb6 but not the 0.3 mg/kg dose blocked fibrosis. However the trend to higher mortality with treatment with the higher dose antibody (10 mg/kg), if confirmed in further experiments, may indicate that partial rather than high-level inhibition of avb6-mediated TGFb activation may yield optimal results. These results support further investigation into blockade of avb6 as a therapeutic modality.
2409
Glycogen Synthase Kinase 3 Participates in Function of Vascular Endothelium
K.C. Cuneo, E. Yazlovitskaya, D. Thotala, D.E. Hallahan Radiation Oncology, Vanderbilt University, Nashville, TN Purpose/Objective: Akt signaling participates in angiogenesis in response to VEGF stimulation of endothelial cells and regulates capillary-like tubule formation which requires the inactivation of GSK3b. Over-expression of GSK-3 induces apoptosis in various cells in culture, and specific inhibitors of GSK-3 ameliorate this apoptotic process. We therefore studied the role of Akt/GSK3 signaling in irradiated vascular endothelium. Materials/Methods: Radiation-induced phosphorylation of Akt and GSK3 was studied in endothelial cells irradiated with 3 Gy. Total protein was extracted and Western immunoblots were probed with antibodies specific for phosphorylated Akt and total Akt. To determine whether Akt participates in GSK3 phosphorylation following irradiation, the dominant negative Akt genetic construct (Ad.⌬ Akt) was overexpressed in primary culture vascular endothelial cells. Western blot analysis of caspase 3 activation was utilized to verify the induction of apoptosis. Capillary tubule formation in matrigel was studied in irradiated vascular endothelium overexpressing Ad.⌬ Akt. Results: Phosphorylation of Akt was minimal in untreated control endothelial cells, but increased within 5 minutes and was maximal at 15 min. Because GSK3b is one of the key effectors of Akt signaling, radiation may also induce phosphorylation of GSK3b. Similarly, minimal phosphorylation of GSK3b was found in untreated control cells, but increased at 5 minutes and was maximal at 15 min. The dominant negative Akt genetic construct (Ad.⌬ AKT) was overexpressed in endothelial cells. Overexpression of Ad.⌬ AKT abrogated GSK3 phosphorylation following irradiation. In comparison, cells transduced with control vector Ad.GFP showed no interruption of radiation-induced GSK3 phosphorylation. 5% of primary culture endothelial cells undergo apoptosis following treatment with Ad.⌬ AKT as compared to 2.5% in the control cells. In comparison, when irradiated, Ad.⌬ AKT transduced cells had a markedly increased percentage of apoptotic cells to 25%, which was significantly increased as compared to treatment with Ad.⌬ AKT or radiation alone (p⬍ 0.001). Caspase 3 cleavage was induced in HUVEC transduced with Ad.⌬ AKT followed by irradiation. In comparison, Ad.⌬ Akt or 3 Gy resulted in minimal caspase 3 cleavage in endothelial cells. We next examined caspase 3 cleavage and propidium iodide staining, which showed that GSK3b dominant negative prevented apoptosis. Dominant negative GSK3b transduced cells were then treated with Akt inhibitor and radiation, which decreased the caspase 3 activity in irradiated endothelial cells treated with Akt inhibitor. In comparison, cells treated with the Ad.GFP control vector showed increased caspase 3 cleavage following treatment with Akt inhibitor and radiation. To verify the role of GSK3b signaling during in vivo microvascular response, we studied capillary formation in the irradiated mouse utilizing VEGF within matrigel implanted subcutaneously into the mouse flank. The mouse flank was then irradiated with 3 Gy and FITC-dextran was injected by tail vein 72 hours later. The FITC-dextran containing capillaries within the Matrigel plug were imaged by fluorescence microscopy. Capillary formation within Matrigel occurred within the irradiated
Proceedings of the 47th Annual ASTRO Meeting
mouse flank. Ad.⌬ Akt and Ad.⌬ GSK3b were added to Matrigel and capillaries were counted by microscopy. The Akt dominant negative entirely eliminated capillary-like tubule formation. In comparison, the GSK3b dominant negative reversed the effect of Akt mutant on capillary formation (P⫽0.04). Conclusions: These data indicate that ionizing radiation-induced Akt signaling contributes to inactivation of GSK3b through phosphorylation of this kinase. GSK3 phosphorylation, in turn, impacts upon endothelial cell survival and capillary formation in mouse models.
2410
Gene Selection by a Genomic Radiation Sensitivity Classifier is Influenced by the Presence of a Mutated ras Gene
J.F. Torres-Roca, S. Eschrich, T. Yeatman Interdisciplinary Oncology, H Lee Moffitt Cancer Center, Tampa, FL Purpose/Objective: In previous studies, we demonstrated the development of a gene expression radiation sensitivity predictor in tumor cell lines, that accurately predicted the survival fraction at 2 Gy (SF2) in 22 of 35 cell lines from the NCI panel of 60. This was accomplished using baseline gene expression profiles derived from microarrays (Affymetrix HU-6800) that were available in the literature. In the classifier design, we treated radiation sensitivity as a continuous variable, and included a multivariate linear regression model as the predictive step. A leave-one-out cross validation model was used in the design. This analytical approach was validated biologically, by demonstrating that one of the selected predictive genes (RbAp48) was involved mechanistically in radiation response. In this work, we sought to determine the role that a mutated ras gene would convey on the baseline gene expression signature and how this would affect the process of radiation sensitivity prediction. Materials/Methods: A total of 48 cell lines from the NCI-panel of 60 were used for this study. SF2s were either obtained from the literature (23 cell lines) or determined in our laboratory using clonogenic assays (25 cell lines). Gene expression profiles were from Affymetrix HU-6800 (7,129 genes) from a previously published study. The gene expression data was preprocessed using the Affymetrix MAS 4.0 algorithm in average difference units. Negative expression values were set to zero and the chips were normalized to the same mean intensity. Ras status (mutated vs. wild-type) was obtained from the literature. Gene selection was done using SAM analysis. Radiation sensitivity (defined as SF2) was treated as a continuous variable. A leave-one-out cross-validation model was used in the classifier design. A multivariate linear regression model was used for radiosensitivity prediction. Results: We initially determined whether Ras status would convey a different baseline gene expression signature. Indeed, SAM analysis identified a subset of genes whose baseline expression separated cells lines by Ras status. Since baseline gene expression profiles were affected by the presence of a mutated Ras protein, we determined whether the genes selected by the radiation sensitivity classifier, were dependent or independent of Ras status. We stratified the original classifier by Ras status, and performed the gene selection step in each group of cell lines. Interestingly, the genes selected by each group were completely different. The three known genes (RbAp48, RSG-19, R5PIA) that had been selected by the overall classifier without Ras stratification and that were previously shown to be highly useful in predicting radiosensitivity, were highly ranked by the Ras mutated cell lines but not by the wild type lines, suggesting that the Ras mutated cell lines were driving the classification process. RbAp48, RSG-19 and R5PIA were ranked 19th, 46th and 262nd out of 7,129 genes by the Ras-mutated cell lines. In contrast wild-type cell lines ranked these genes 743rd, 758th and 397th respectively. When all cell lines were taken together, these three genes ranked in the top 10 genes (5th, 1st and 9th). Conclusions: The process of gene selection by a biologically-validated genomic radiation sensitivity classifier, is influenced by the presence of a mutated Ras protein. The translation of genomic predictive models to the clinic should take this observation into account.
2411
Non-Invasive Monitoring of Radiation Induced Liver Damage Using Spectroscopic Imaging
31
P Magnetic Resonance
C. Landis,2 L. Liu,1 D. Shafritz,2 J. Roy-Chowdhury,2 H. Hetherington,2 C. Guha1 1 Radiation Oncology, Montefiore Medical Center - Albert Einstein College of Medicine, Bronx, NY, 2Marion Bessin Liver Research Center, Bronx, NY Purpose/Objective: The combination of partial hepatectomy (PH) and whole liver irradiation (PHRT) leads to extensive radiation-induced liver damage (RILD) with 70 – 80% mortality in rats (1). Furthermore, hepatocyte transplantation ameliorated RILD in this model. We used 31P Magnetic Resonance Spectroscopic Imaging (MRSI) to non-invasively monitor changes in phosphorylated metabolites following PHRT as prognostic markers for RILD. Materials/Methods: F344 rats were subjected to 70% PH alone or PH followed by hepatic RT (50 Gy). For MR studies, a 9.4 T Varian INOVA MR system with a birdcage coil for 1H MRI and a surface coil for 31P MRS were used. MR acquisition was gated to the mechanical ventilation rate. A standard multi-slice spin echo imaging sequence was used for scout images (TE⫽25ms, TR⫽1s, FOV⫽64⫻64mm, ns⫽17). For 31P MRSI, a 45° nonselective excitation and 3D phase encoding using a spherical k-space sampling scheme (FOV⫽64⫻64x64mm, TR⫽1s). Data was processed using routines written in the MATLAB programming language. MRS studies were performed 1, 3, and 8 days after PH or PHRT (n⫽3– 4 rats/time point). Livers from sacrificed animals were analyzed for hepatocyte regeneration. Results: Compared to untreated controls (1.32 ⫾ 0.09), the ATP/Pi peak area ratios were significantly reduced in PH (0.87 ⫾ 0.11) and PHRT (0.70 ⫾ 0.04) groups at 1d. BrdU incorporation was markedly reduced in the PHRT group compared to PH alone. A representative MRI scout image and 31P MR spectra from 8 days after a) PH alone or b) PHRT is shown. The spectra are derived from MRSI data within the indicated region of interest. The peak assignments for various phosphorylated metabolites are also indicated. At 8 days, the PH only group exhibits a normalized ATP/Pi ratio (1.28 ⫾ 0.14), indistinguishable from untreated controls. In contrast, the ATP/Pi ratio did not return to baseline in the PHRT rats (1.03 ⫾ 0.07, p⬍0.03).
S473