- Email: [email protected]
Effect of difluoromethylornithine, a chemotherapeutic agent, on wound healing
308
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
ited SNAP-mediated PGP upregulation. Rhodamine transport was increased by cytomix and SNAP, but blocked by LY (prelim. data). Conclusion: PGP expression and function in enterocytes is upregulated by proinflammatory cytokines and NO in a PI3K dependent manner. This effect may be part of an adaptive mechanism to limit epithelial injury in response to inflammatory conditions. P113. Microarray Analysis of Regional Dura Mater and Identification of a Transcription Factor Regulating Epithelial to Mesenchymal Cell Transitions. R. P. Nacamuli, M.D., K. D. Fong, M.D., H. M. Song, M.D., T. D. Fang, M.D., A. Salim, M.D., Y. Shi, B.S., M. T. Longaker, M.D. Stanford University School of Medicine. Introduction: Although a wealth of data demonstrates that regionally patterned dura mater regulates suture fusion or patency, the onset and extent of this patterning remains unknown. In this study we used microarray analyses as a starting point to investigate the differences in suture-specific dura mater from the mouse posterior frontal (PF) and sagittal (SAG) suture. Methods: Regional dura mater was harvested from the PF and SAsutures of 15 and 25 day male CD-1 mice (n ⫽ 40 each) and RNA isolated. Microarray analyses were performed using 42,000 element cDNA arrays. Slug was also evaluated in non-suture dura from young (5-day-old) and adult (60-day-old) animals. Investigation into regulation of novel genes, such as the transcription factor Slug, were carried out using rat calvarial osteoblasts stimulated with FGF-2, TGF-beta 1, and BMP2 and 4. Results: Microarray analyses demonstrated differential regulation of over 220 genes in the PF suture at d15, and over 280 genes at d25. Although the number of genes down-regulated was similar, there was a dramatic increase in the number of genes up-regulated in the PF suture at d25 vs. d15 (137 vs. 43). Distinct patterns of expression were seen for several groups of genes, including cytokines (FGF and FGF receptors) and extra-cellular matrix molecules (Col-1, osteopontin, osteocalcin). Several novel genes were identified, including the transcription factor Slug. QRT-PCR verified a ⬎ 4-fold increase in Slug mRNA in PF vs. SAG dura, and a ⬎ 3-fold increase in young vs. adult dura mater. Functional studies demonstrated a selective inhibition of Slug mRNA in osteoblasts by FGF-2 and TGF-beta 1, but not the BMPs, up to 24 hrs post stimulation (see figure). Conclusions: Our data demonstrate genomic patterning of regional dura mater as early as ten days prior to PF suture fusion. In addition we show that the transcription factor Slug, an evolutionarily conserved gene involved in epithelial-to-mesenchymal cell transitions, is regulated by two growth factors known to be expressed in dura mater. P114. Inhibition of the PI3K/AKT Pathway Enhances Trail Induced Apoptosis in Neuroblastoma Cells. P. A. Efron, M.D., M. K. Chen, M.D., W. Dai, M.D., M. R. Langham Jr., M.D., E. A. Beierle, M.D. University of Florida Department of Surgery, Gainesville, FL. Purpose: The binding of TRAIL to membrane receptors activates caspase 8, which initiates the extrinsic apoptotic pathway. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been demonstrated to protect a number of human tumor cells from apoptosis that may occur via both the extrinsic and intrinsic apoptotic pathways. Investigators have shown that neuroblastoma are resistant to TRAIL induced apoptosis. We hypothesize that inhibition of the PI3K/Akt pathway may enhance the ability of TRAIL to induce apoptosis in neuroblastoma cells. Methods: Human neuroblastoma cells (IMR-32) were grown to 80% confluence. Cells were then divided into four groups receiving the following treatment over 48 hr: (Control) standard growth media, (LY) standard media with 20 g/mL LY294002 (a specific PI3K inhibitor), (TRAIL) standard media with 100 ng/mL TRAIL, (LY&TRAIL) standard media with 20 g/mL LY294002 ⫻ 24 hr followed by 100 ng/mL
TRAIL ⫻ 24 hr. Flow cytometry was used to assess cell viability (7-AAD), apoptosis (Annexin V), mitochondrial transmembrane potential/apoptosis (DioC6), and active caspases 8 (extrinsic pathway) and 9 (intrinsic pathway). Results: There was no difference between the numbers of apoptotic and dead cells in control IMR-32 cells compared to those treated with TRAIL. LY294002 alone also did not increase apoptosis or cell death. The mitochondrial transmembrane potential was decreased 25% in the group pre-treated with LY294002 (LY&TRAIL) compared to control cells. Activity of caspase 8 increased by 10.5%, and activity of caspase 9 increased by 16% in the LY&TRAIL treated cells compared to controls. LY&TRAIL resulted in increased levels of TRAIL induced apoptosis by 5.5% and cell death by 9.5% compared to control cells. Conclusions: The resistance of IMR-32 neuroblastoma cells to TRAIL induced apoptosis is partly dependent upon the activation of the PI3K/Akt pathway. This protection appears to occur through inhibition of both the extrinsic and intrinsic apoptotic pathways. Specific blockage of the PI3K/Akt pathway can enhance the capacity for TRAIL induced apoptosis of neuroblastoma cells. P115. Does Gender Influence Outcome in Pediatric Trauma? B. A. Gaines, M.D., L. Cassidy, Ph.D., H. R. Ford, M.D. University of Pittsburgh, Division of Pediatric Surgery, Pittsburgh, PA. Introduction: Recent experimental data suggests that sex hormones play an important role in host response to injury. Studies on the influence of gender on outcome in adult trauma populations have yielded mixed results, with some demonstrating no difference between men and women and others showing a slight advantage to female gender. To date, there have been no studies evaluating the role of gender in pediatric trauma patients. We hypothesize that since the hormonal milieu in pre-pubertal children is the same in both boys and girls, there should be no gender-related differences in outcome between them. Furthermore, as children progress through puberty, any role that sex-hormones play in outcome should become apparent. Methods: A five-year (1994 –1999) retrospective review of the Pennsylvania Trauma Outcome Study database was performed and information regarding all children ⱕ16 years was abstracted. We evaluated the effects of age (ⱕ10 years and 11–16 years), gender, injury severity (measured by Injury Severity Score, ISS), and type of injury on mortality. Statistical analysis was performed using chisquare and multivariate logistic regression (Stata, College Station, Texas). P ⬍ 0.05 was considered statistically significant. Results: 17,690 children were admitted to Pennsylvania trauma centers during the study period; 67% were male and the overall mortality was 3.8%. There was no gender-related difference in mortality in children ⱕ 10 years of age with mild (ISS ⱕ 15; p ⫽ .4), moderate (ISS 16 –24; p ⫽ .9), or severe (ISS ⬎ 25; p ⫽ .5) injuries, head injuries (p ⫽ .5), or injury to the trunk (p ⫽ .6) or extremities (p ⫽ .4). In contrast, girls 11–16 years of age with a head injury had a 3 times higher risk of mortality than boys (OR 3.09, 95% CI 2.63–3.64) despite equivalent admission GCS and ISS. Conclusion: As expected, gender does not influence mortality in children ⱕ 10 years of age. On the other hand, adolescent girls appear to have a higher than expected mortality from head injuries. Whether this is a result of hormonal influences or other factors have yet to be determined.
TABLE—ABSTRACT P115 Head injury ⱕ10 years ⬎10 years
Male (died/total)
Female (died/total)
P-value
151/2593 118/1847
96/1385 62/712
0.4 0.04
P116. Effect of Difluoromethylornithine, a Chemotherapeutic Agent, on Wound Healing. V. Ahuja, M.D.,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS J. M. Abrams, M.D., U. Tantry, Ph.D., J. Park, M.D., A. Barbul, M.D. Sinai Hospital. Introduction: Difluoromethylornithine (DFMO) is a potential chemotherapeutic agent and currently in Phase II trials for colon and prostate malignancies. DFMO will potentially be used for chemotherapy in postoperative patients in low dosage. Therefore, we studied the effect of low dosage DFMO on wound healing. Methods: Two groups of twenty male Sprague Dawley rats underwent a midline dorsal incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. In Exp 1, ten rats were injected daily with DMFO (500 mg/kg IP) starting on day of wounding while controls (ten rats) received equivalent volume of normal saline. In Exp 2, DFMO and normal saline treatments were delayed until postwounding day 3. All rats were sacrificed on day 10 post-wounding. Incisional wound breaking strength (WBS, g) and sponge hydroxyproline (OHP, g/100mg sponge) content (collagen deposition), were assessed. Efficacy of DFMO treatment was assessed by measuring wound fluid polyamines, putrescine (PUT) and spermidine (SPER)(M) Results: Treatment with DFMO starting on day 0 (Exp 1) resulted in a significant decrease in collagen deposition and wound breaking strength. Neither of the wound healing parameters was affected when treatment was begun 3 days post-wounding (Exp 2). Conclusion: Wound healing is decreased when DFMO treatment is started on the day of surgery. This suggests that DFMO treatment may play a critical role in the synthesis of collagen in early wound healing and chemotherapy with DFMO should be delayed for patients requiring surgery.
TABLE—ABSTRACT P116 Group EXP 1 Control DFMO EXP 2 Control DFMO
WBS
OHP
PUT
SPER
409 ⫾ 82 340 ⫾ 41*
1435 ⫾ 102 1014 ⫾ 67**
11.6 ⫾ 3.4 2.2 ⫾ 0.4**
ND ND
376 ⫾ 94 354 ⫾ 41
1568 ⫾ 90 1624 ⫾ 150
15.3 ⫾ 5.2 5.7 ⫾ 5.4**
95.4 ⫾ 18.6 63.1 ⫾ 12.8**
Mean ⫾ standard deviation. *P ⬍ 0.05 and **P ⬍ 0.005 by t-test; Student’s t-test; ND—not determined.
P117. FAK Expression in Neuroblastoma. E. A. Beierle, M.D., W. Dai, M.D., W. G. Cance, M.D., M. R. Langham Jr., M.D., M. K. Chen, M.D. University of Florida Department of Surgery, Gainesville, FL. Purpose: Focal adhesion kinase (FAK) is an anti-apoptotic protein tyrosine kinase that is over expressed in many human tumors, but it has not been well characterized in neuroblastoma. Previous studies have shown that neuroblastoma cells cultured with VEGF or in coculture with hepatocytes are resistant to apoptotic stimuli. We hypothesize that anti-apoptotic FAK is present in neuroblastoma cells and its expression will be upregulated when the cells are exposed to VEGF or when cocultured with hepatocytes. Methods: Human neuroblastoma cells (IMR-32) are cultured under three conditions. 1) Standard media (control). 2) Media ⫹ VEGF (5, 20, 50, 100
309
ng/mL). 3) Cocultured with hepatocytes (6, 12, 24 hours). FAK expression is measured by Western blot. Experiments are repeated in triplicate to assure consistency in results. Results: Representative Western blots are presented below. FAK protein expression is increased with increasing amounts of VEGF in the culture media. This finding becomes apparent after the addition of 20ng/mL VEGF and continues up to 100 ng/mL. FAK expression is also increased in IMR-32 neuroblastoma cells that are grown with hepatocytes in coculture when compared to control cells. The increase in FAK in cocultured cells is seen as early as 6 hours. Conclusion: Neuroblastoma cells cultured with the addition of VEGF or in coculture have been shown to have decreased apoptosis. In this study, these culture conditions also lead to an increase in the expression of FAK. FAK may play a role in the regulation of apoptosis in neuroblastoma.
P118. Changes in Oxygen Tension Modulate Osteoblast Gene Expression and Long-term Differentiation. A. Salim, M.D., R. P. Nacamuli, M.D., A. J. Giaccia, Ph.D., M. T. Longaker, M.D. Stanford University. Introduction: Disruption of blood flow with bony and soft-tissue injury results in a hypoxic gradient within the wound microenvironment. As a first step to understanding osteoblast behavior following hypoxic insult, we perform large-scale transcriptional profiling of hypoxic and anoxic osteoblasts. In addition, we investigate long-term osteoblast differentiation after brief hypoxic exposure. Methods: RNA from murine calvarial osteoblasts cultured in 21%, 2%, or 0% O2 for up to 24 hours was used for microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Osteoblasts exposed to 24 hours of hypoxia were differentiated for 28 days and compared to non-hypoxic cultures via qRT-PCR and bone-nodule staining by the Von Kossa method. Results: Osteoblast exposure to 0% or 2% O2 for 24 hours resulted in statistically significant expression changes for approximately 700 genes in each group. Of these, only 89 genes were shared by both groups. We observed differential regulation of osteogenic and angiogenic markers, transcription and growth factors, and inflammatory signaling modulators. As expected, both conditions induced anaerobic glycolysis, but only anoxia elevated heat-shock response elements. Surprisingly, osteoblasts exposed to 24 hours of anoxia demonstrated diminished bone nodule formation at 28 days. Conclusions: Gene expression changes in osteoblasts within the hypoxic post-injury microenvironment remain largely unknown. Transcriptional profiling reveals distinct patterns of expression at 0% versus 2% O2 relative to ambient oxygen. Furthermore, in vitro osteoblast differentiation is diminished even after brief anoxic insult. Together with induction of angiogenic and inflammatory genes, this underscores the involvement of additional cell types for successful bone repair.
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
ited SNAP-mediated PGP upregulation. Rhodamine transport was increased by cytomix and SNAP, but blocked by LY (prelim. data). Conclusion: PGP expression and function in enterocytes is upregulated by proinflammatory cytokines and NO in a PI3K dependent manner. This effect may be part of an adaptive mechanism to limit epithelial injury in response to inflammatory conditions. P113. Microarray Analysis of Regional Dura Mater and Identification of a Transcription Factor Regulating Epithelial to Mesenchymal Cell Transitions. R. P. Nacamuli, M.D., K. D. Fong, M.D., H. M. Song, M.D., T. D. Fang, M.D., A. Salim, M.D., Y. Shi, B.S., M. T. Longaker, M.D. Stanford University School of Medicine. Introduction: Although a wealth of data demonstrates that regionally patterned dura mater regulates suture fusion or patency, the onset and extent of this patterning remains unknown. In this study we used microarray analyses as a starting point to investigate the differences in suture-specific dura mater from the mouse posterior frontal (PF) and sagittal (SAG) suture. Methods: Regional dura mater was harvested from the PF and SAsutures of 15 and 25 day male CD-1 mice (n ⫽ 40 each) and RNA isolated. Microarray analyses were performed using 42,000 element cDNA arrays. Slug was also evaluated in non-suture dura from young (5-day-old) and adult (60-day-old) animals. Investigation into regulation of novel genes, such as the transcription factor Slug, were carried out using rat calvarial osteoblasts stimulated with FGF-2, TGF-beta 1, and BMP2 and 4. Results: Microarray analyses demonstrated differential regulation of over 220 genes in the PF suture at d15, and over 280 genes at d25. Although the number of genes down-regulated was similar, there was a dramatic increase in the number of genes up-regulated in the PF suture at d25 vs. d15 (137 vs. 43). Distinct patterns of expression were seen for several groups of genes, including cytokines (FGF and FGF receptors) and extra-cellular matrix molecules (Col-1, osteopontin, osteocalcin). Several novel genes were identified, including the transcription factor Slug. QRT-PCR verified a ⬎ 4-fold increase in Slug mRNA in PF vs. SAG dura, and a ⬎ 3-fold increase in young vs. adult dura mater. Functional studies demonstrated a selective inhibition of Slug mRNA in osteoblasts by FGF-2 and TGF-beta 1, but not the BMPs, up to 24 hrs post stimulation (see figure). Conclusions: Our data demonstrate genomic patterning of regional dura mater as early as ten days prior to PF suture fusion. In addition we show that the transcription factor Slug, an evolutionarily conserved gene involved in epithelial-to-mesenchymal cell transitions, is regulated by two growth factors known to be expressed in dura mater. P114. Inhibition of the PI3K/AKT Pathway Enhances Trail Induced Apoptosis in Neuroblastoma Cells. P. A. Efron, M.D., M. K. Chen, M.D., W. Dai, M.D., M. R. Langham Jr., M.D., E. A. Beierle, M.D. University of Florida Department of Surgery, Gainesville, FL. Purpose: The binding of TRAIL to membrane receptors activates caspase 8, which initiates the extrinsic apoptotic pathway. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been demonstrated to protect a number of human tumor cells from apoptosis that may occur via both the extrinsic and intrinsic apoptotic pathways. Investigators have shown that neuroblastoma are resistant to TRAIL induced apoptosis. We hypothesize that inhibition of the PI3K/Akt pathway may enhance the ability of TRAIL to induce apoptosis in neuroblastoma cells. Methods: Human neuroblastoma cells (IMR-32) were grown to 80% confluence. Cells were then divided into four groups receiving the following treatment over 48 hr: (Control) standard growth media, (LY) standard media with 20 g/mL LY294002 (a specific PI3K inhibitor), (TRAIL) standard media with 100 ng/mL TRAIL, (LY&TRAIL) standard media with 20 g/mL LY294002 ⫻ 24 hr followed by 100 ng/mL
TRAIL ⫻ 24 hr. Flow cytometry was used to assess cell viability (7-AAD), apoptosis (Annexin V), mitochondrial transmembrane potential/apoptosis (DioC6), and active caspases 8 (extrinsic pathway) and 9 (intrinsic pathway). Results: There was no difference between the numbers of apoptotic and dead cells in control IMR-32 cells compared to those treated with TRAIL. LY294002 alone also did not increase apoptosis or cell death. The mitochondrial transmembrane potential was decreased 25% in the group pre-treated with LY294002 (LY&TRAIL) compared to control cells. Activity of caspase 8 increased by 10.5%, and activity of caspase 9 increased by 16% in the LY&TRAIL treated cells compared to controls. LY&TRAIL resulted in increased levels of TRAIL induced apoptosis by 5.5% and cell death by 9.5% compared to control cells. Conclusions: The resistance of IMR-32 neuroblastoma cells to TRAIL induced apoptosis is partly dependent upon the activation of the PI3K/Akt pathway. This protection appears to occur through inhibition of both the extrinsic and intrinsic apoptotic pathways. Specific blockage of the PI3K/Akt pathway can enhance the capacity for TRAIL induced apoptosis of neuroblastoma cells. P115. Does Gender Influence Outcome in Pediatric Trauma? B. A. Gaines, M.D., L. Cassidy, Ph.D., H. R. Ford, M.D. University of Pittsburgh, Division of Pediatric Surgery, Pittsburgh, PA. Introduction: Recent experimental data suggests that sex hormones play an important role in host response to injury. Studies on the influence of gender on outcome in adult trauma populations have yielded mixed results, with some demonstrating no difference between men and women and others showing a slight advantage to female gender. To date, there have been no studies evaluating the role of gender in pediatric trauma patients. We hypothesize that since the hormonal milieu in pre-pubertal children is the same in both boys and girls, there should be no gender-related differences in outcome between them. Furthermore, as children progress through puberty, any role that sex-hormones play in outcome should become apparent. Methods: A five-year (1994 –1999) retrospective review of the Pennsylvania Trauma Outcome Study database was performed and information regarding all children ⱕ16 years was abstracted. We evaluated the effects of age (ⱕ10 years and 11–16 years), gender, injury severity (measured by Injury Severity Score, ISS), and type of injury on mortality. Statistical analysis was performed using chisquare and multivariate logistic regression (Stata, College Station, Texas). P ⬍ 0.05 was considered statistically significant. Results: 17,690 children were admitted to Pennsylvania trauma centers during the study period; 67% were male and the overall mortality was 3.8%. There was no gender-related difference in mortality in children ⱕ 10 years of age with mild (ISS ⱕ 15; p ⫽ .4), moderate (ISS 16 –24; p ⫽ .9), or severe (ISS ⬎ 25; p ⫽ .5) injuries, head injuries (p ⫽ .5), or injury to the trunk (p ⫽ .6) or extremities (p ⫽ .4). In contrast, girls 11–16 years of age with a head injury had a 3 times higher risk of mortality than boys (OR 3.09, 95% CI 2.63–3.64) despite equivalent admission GCS and ISS. Conclusion: As expected, gender does not influence mortality in children ⱕ 10 years of age. On the other hand, adolescent girls appear to have a higher than expected mortality from head injuries. Whether this is a result of hormonal influences or other factors have yet to be determined.
TABLE—ABSTRACT P115 Head injury ⱕ10 years ⬎10 years
Male (died/total)
Female (died/total)
P-value
151/2593 118/1847
96/1385 62/712
0.4 0.04
P116. Effect of Difluoromethylornithine, a Chemotherapeutic Agent, on Wound Healing. V. Ahuja, M.D.,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS J. M. Abrams, M.D., U. Tantry, Ph.D., J. Park, M.D., A. Barbul, M.D. Sinai Hospital. Introduction: Difluoromethylornithine (DFMO) is a potential chemotherapeutic agent and currently in Phase II trials for colon and prostate malignancies. DFMO will potentially be used for chemotherapy in postoperative patients in low dosage. Therefore, we studied the effect of low dosage DFMO on wound healing. Methods: Two groups of twenty male Sprague Dawley rats underwent a midline dorsal incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. In Exp 1, ten rats were injected daily with DMFO (500 mg/kg IP) starting on day of wounding while controls (ten rats) received equivalent volume of normal saline. In Exp 2, DFMO and normal saline treatments were delayed until postwounding day 3. All rats were sacrificed on day 10 post-wounding. Incisional wound breaking strength (WBS, g) and sponge hydroxyproline (OHP, g/100mg sponge) content (collagen deposition), were assessed. Efficacy of DFMO treatment was assessed by measuring wound fluid polyamines, putrescine (PUT) and spermidine (SPER)(M) Results: Treatment with DFMO starting on day 0 (Exp 1) resulted in a significant decrease in collagen deposition and wound breaking strength. Neither of the wound healing parameters was affected when treatment was begun 3 days post-wounding (Exp 2). Conclusion: Wound healing is decreased when DFMO treatment is started on the day of surgery. This suggests that DFMO treatment may play a critical role in the synthesis of collagen in early wound healing and chemotherapy with DFMO should be delayed for patients requiring surgery.
TABLE—ABSTRACT P116 Group EXP 1 Control DFMO EXP 2 Control DFMO
WBS
OHP
PUT
SPER
409 ⫾ 82 340 ⫾ 41*
1435 ⫾ 102 1014 ⫾ 67**
11.6 ⫾ 3.4 2.2 ⫾ 0.4**
ND ND
376 ⫾ 94 354 ⫾ 41
1568 ⫾ 90 1624 ⫾ 150
15.3 ⫾ 5.2 5.7 ⫾ 5.4**
95.4 ⫾ 18.6 63.1 ⫾ 12.8**
Mean ⫾ standard deviation. *P ⬍ 0.05 and **P ⬍ 0.005 by t-test; Student’s t-test; ND—not determined.
P117. FAK Expression in Neuroblastoma. E. A. Beierle, M.D., W. Dai, M.D., W. G. Cance, M.D., M. R. Langham Jr., M.D., M. K. Chen, M.D. University of Florida Department of Surgery, Gainesville, FL. Purpose: Focal adhesion kinase (FAK) is an anti-apoptotic protein tyrosine kinase that is over expressed in many human tumors, but it has not been well characterized in neuroblastoma. Previous studies have shown that neuroblastoma cells cultured with VEGF or in coculture with hepatocytes are resistant to apoptotic stimuli. We hypothesize that anti-apoptotic FAK is present in neuroblastoma cells and its expression will be upregulated when the cells are exposed to VEGF or when cocultured with hepatocytes. Methods: Human neuroblastoma cells (IMR-32) are cultured under three conditions. 1) Standard media (control). 2) Media ⫹ VEGF (5, 20, 50, 100
309
ng/mL). 3) Cocultured with hepatocytes (6, 12, 24 hours). FAK expression is measured by Western blot. Experiments are repeated in triplicate to assure consistency in results. Results: Representative Western blots are presented below. FAK protein expression is increased with increasing amounts of VEGF in the culture media. This finding becomes apparent after the addition of 20ng/mL VEGF and continues up to 100 ng/mL. FAK expression is also increased in IMR-32 neuroblastoma cells that are grown with hepatocytes in coculture when compared to control cells. The increase in FAK in cocultured cells is seen as early as 6 hours. Conclusion: Neuroblastoma cells cultured with the addition of VEGF or in coculture have been shown to have decreased apoptosis. In this study, these culture conditions also lead to an increase in the expression of FAK. FAK may play a role in the regulation of apoptosis in neuroblastoma.
P118. Changes in Oxygen Tension Modulate Osteoblast Gene Expression and Long-term Differentiation. A. Salim, M.D., R. P. Nacamuli, M.D., A. J. Giaccia, Ph.D., M. T. Longaker, M.D. Stanford University. Introduction: Disruption of blood flow with bony and soft-tissue injury results in a hypoxic gradient within the wound microenvironment. As a first step to understanding osteoblast behavior following hypoxic insult, we perform large-scale transcriptional profiling of hypoxic and anoxic osteoblasts. In addition, we investigate long-term osteoblast differentiation after brief hypoxic exposure. Methods: RNA from murine calvarial osteoblasts cultured in 21%, 2%, or 0% O2 for up to 24 hours was used for microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Osteoblasts exposed to 24 hours of hypoxia were differentiated for 28 days and compared to non-hypoxic cultures via qRT-PCR and bone-nodule staining by the Von Kossa method. Results: Osteoblast exposure to 0% or 2% O2 for 24 hours resulted in statistically significant expression changes for approximately 700 genes in each group. Of these, only 89 genes were shared by both groups. We observed differential regulation of osteogenic and angiogenic markers, transcription and growth factors, and inflammatory signaling modulators. As expected, both conditions induced anaerobic glycolysis, but only anoxia elevated heat-shock response elements. Surprisingly, osteoblasts exposed to 24 hours of anoxia demonstrated diminished bone nodule formation at 28 days. Conclusions: Gene expression changes in osteoblasts within the hypoxic post-injury microenvironment remain largely unknown. Transcriptional profiling reveals distinct patterns of expression at 0% versus 2% O2 relative to ambient oxygen. Furthermore, in vitro osteoblast differentiation is diminished even after brief anoxic insult. Together with induction of angiogenic and inflammatory genes, this underscores the involvement of additional cell types for successful bone repair.