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Cold Ischemia and Reperfusion Injury in the Steatotic Livery Is Mediated by Acute Endoplasmic Reticuluim Stress
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS MSCs was significantly greater compared to the WT (677.2 þ/- 67.25 vs. 380.5 þ/- 23.29 pg/mL) as well as with TNF stimulation (1415.0 þ/- 81.41 vs. 543.6 þ/- 12.0 pg/mL). Conclusions: Pre-ischemic treatment with TLR4KO MSCs improves myocardial recovery after I/R. The improved recovery seen with TLR4KO MSCs may in part be due to higher levels of basal and stimulated VEGF production. 46.7. Cold Ischemia and Reperfusion Injury in the Steatotic Livery Is Mediated by Acute Endoplasmic Reticuluim Stress. C. D. Anderson, G. Upadhya, K. Conzen, J. Jia, N. Davidson, S. Ramachandran, T. Mohanakumar, W. C. Chapman; Washington University in St.Louis, St. Louis, MO Background: Hepatic steatosis is an increasingly prevalent problem in liver transplantation (LT) leading to increased cold ischemia and reperfusion (CIR) injury and severe allograft dysfunction. Hepatic steatosis is strongly associated with endoplasmic reticulum (ER) stress which can trigger cell death via multiple pathways. We hypothesized that acute ER stress gene expression following CIR injury may mediate injury in steatotic livers following liver transplantation. Methods: Steatotic livers from obese Zucker rats were transplanted into lean recipients (cold preservation-2 hours, reperfusion-12 hr) (n ¼ 5). A cohort (n ¼ 5) of steatotic livers were preserved with HTK þ 10 mM taurine-conjugated ursodeoxycholic acid (TUDCA) a chemical chaperone known to decrease ER stress. TUDCA (0.5 mg/kg IP) or saline was given at the time of reperfusion. ER stress was measured by quantitative real-time PCR (QRTPCR) (GRP-78, ATF4, IRE1a, CHOP) and western analysis (XBP-1 splicing). Proapoptotic Bcl-2 genes (Bax, Bim, Bid) were measured by QRTPCR. Liver injury was measured by serum ALT and histologic characterization. Transplanted lean livers, lean shams, and obese shams served as controls (n ¼ 3 each). Results: Following LT, steatotic allografts had increased ER stress gene expression (p < 0.001 for all) and XPB-1 splicing compared to lean. CHOP, a specific cell death mediator was increased 26 fold. Livers preserved in TUDCA has an ER stress response as demonstrated by decreased gene expression (p < 0.001 for all). In addition, Bax and Bim expression were both decreased in TUDCA stored steatotic allografts (p < 0.001). Histologicaly, TUDCA treatment decreased periportal and lobular graft inflammation and surrogate markers of graft injury were reduced (decreased ALT, p ¼ 0.02). Conclusions: CIR induced acute ER stress is an important mediator of transplant injury steatotic livers. This study suggests that attenuation of ER stress is a potential hepatoprotective strategy for steatotic allografts. In addition, these results suggest that the downstream mediators of CHOP including Bax and Bim may represent important pathways for mechanistic investigation of this protection.
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Introduction: Stem cell function is closely related to gene expression. Hematopoietic stem cells (HSCs) are the most widely studied adult stem cell population and as such, provide an ideal model for defining stem cell transcriptional identity. Although HSCs can be sorted by FACS into tight populations using surface antigen expression, several investigators have demonstrated heterogeneity of function within these seemingly homogenous HSC populations. To date, the nature of this heterogeneity has been unclear, largely due to the absence of single cell analysis to precisely define the HSC expression within a given hierarchical category. We utilized a novel high throughput single cell transcriptional analysis to systematically characterize the transcriptional profiles of individual HSCs. Methods: We harvested total bone marrow from C57BL/6 mice and performed an immunodepletion of lineage-committed cells using magnetic microbeads. Using fluorescence activated cell sorting (FACS), we sorted individual HSCs from the CD34high and CD34low fractions of lineageneg/Sca-1þ/cKitþ[LSK]/CD150þ/ CD48-/CD135- bone marrow cells into each well of 96-well plates that had been preloaded with qRT-PCR reagents. We performed low cycle pre-amplification to create cDNA across 48 discrete gene targets for each single cell. Then, using a microfluidic chip-based system, we ran simultaneous real-time quantitative PCR on each single cell isolate across each of the 48 genes of interest, yielding 9,216 individual data points for analysis. The data was analyzed using a clustering algorithm based on the mathematical principles of fuzzy set theory to compare gene expression across all cells. Results: We analyzed the expression of 48 genes across 190 individual HSCs. All cells expressed cKit, Sca-1, and CD45, as well as the stem cell genes TERT, REST, and cMyc, confirming their identity as HSCs. By comparing single cells from the CD34high and CD34low fractions of HSCs, we were able to define unique subpopulations, or clusters, within these tightly sorted populations. The two cell populations had differential membership in the clusters, consistent with their known functional differences. The CD34low group had a higher percentage of cells with a largely quiescent transcriptional program, whereas the CD34high cells were more actively cycling. Further, the variability of gene expression within each subpopulation demonstrated significant heterogeneity. Conclusion: Microfluidic largescale integration single cell gene expression analysis reveals significant transcriptional heterogeneity within two tightly sorted LTHSC populations. This demonstrates that surface antigen expression alone is insufficient to define stem cell functional groups and suggests that cells with similar surface marker profiles exhibit heterogeneous transcriptional programs. In aggregate, these results provide a novel paradigm for understanding HSC organization as a heterogeneous group of cells manifested through multiple transcriptionally defined subpopulations, each containing transcriptionally variable cells.
TRAUMA AND CRITICAL CARE 4: ISCHEMIA/ REPERFUSION 47.1. Hepatocyte Interferon Regulatory Factor-1 Mediates Acetylation of High Mobility Group Box-1 to Promote Release during Ischemic Liver Injury. R. Dhupar, J. Klune, J. Evankovich, S. W. Cho, J. Cardinal, T. R. Billiar, D. A. Geller, A. Tsung; University of Pittsburgh Medical Center, Pittsburgh, PA
46.8. Single Cell Analysis Demonstrates Significant Transcriptional Heterogeneity Within Tightly-Sorted Murine Hematopoietic Stem Cell Populations. J. P. Glotzbach, I. N. Vial, M. Januszyk, V. W. Wong, H. Thangarajah, M. T. Longaker, G. C. Gurtner; Stanford University, Stanford, CA
Background: Danger associated molecular patterns (DAMPs) initiate inflammatory pathways that are common to both sterile and infectious processes. One such DAMP, high molecular group box-1 (HMGB-1), is a nuclear protein that is released from stressed hepatocytes and mediates inflammation after liver ischemia/reperfusion (I/R) injury. We sought to determine the role of the transcription factor, Interferon Regulatory Factor-1 (IRF-1), in the mechanism of HMGB-1 release following ischemic liver injury. Methods: For in
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Introduction: Stem cell function is closely related to gene expression. Hematopoietic stem cells (HSCs) are the most widely studied adult stem cell population and as such, provide an ideal model for defining stem cell transcriptional identity. Although HSCs can be sorted by FACS into tight populations using surface antigen expression, several investigators have demonstrated heterogeneity of function within these seemingly homogenous HSC populations. To date, the nature of this heterogeneity has been unclear, largely due to the absence of single cell analysis to precisely define the HSC expression within a given hierarchical category. We utilized a novel high throughput single cell transcriptional analysis to systematically characterize the transcriptional profiles of individual HSCs. Methods: We harvested total bone marrow from C57BL/6 mice and performed an immunodepletion of lineage-committed cells using magnetic microbeads. Using fluorescence activated cell sorting (FACS), we sorted individual HSCs from the CD34high and CD34low fractions of lineageneg/Sca-1þ/cKitþ[LSK]/CD150þ/ CD48-/CD135- bone marrow cells into each well of 96-well plates that had been preloaded with qRT-PCR reagents. We performed low cycle pre-amplification to create cDNA across 48 discrete gene targets for each single cell. Then, using a microfluidic chip-based system, we ran simultaneous real-time quantitative PCR on each single cell isolate across each of the 48 genes of interest, yielding 9,216 individual data points for analysis. The data was analyzed using a clustering algorithm based on the mathematical principles of fuzzy set theory to compare gene expression across all cells. Results: We analyzed the expression of 48 genes across 190 individual HSCs. All cells expressed cKit, Sca-1, and CD45, as well as the stem cell genes TERT, REST, and cMyc, confirming their identity as HSCs. By comparing single cells from the CD34high and CD34low fractions of HSCs, we were able to define unique subpopulations, or clusters, within these tightly sorted populations. The two cell populations had differential membership in the clusters, consistent with their known functional differences. The CD34low group had a higher percentage of cells with a largely quiescent transcriptional program, whereas the CD34high cells were more actively cycling. Further, the variability of gene expression within each subpopulation demonstrated significant heterogeneity. Conclusion: Microfluidic largescale integration single cell gene expression analysis reveals significant transcriptional heterogeneity within two tightly sorted LTHSC populations. This demonstrates that surface antigen expression alone is insufficient to define stem cell functional groups and suggests that cells with similar surface marker profiles exhibit heterogeneous transcriptional programs. In aggregate, these results provide a novel paradigm for understanding HSC organization as a heterogeneous group of cells manifested through multiple transcriptionally defined subpopulations, each containing transcriptionally variable cells.
TRAUMA AND CRITICAL CARE 4: ISCHEMIA/ REPERFUSION 47.1. Hepatocyte Interferon Regulatory Factor-1 Mediates Acetylation of High Mobility Group Box-1 to Promote Release during Ischemic Liver Injury. R. Dhupar, J. Klune, J. Evankovich, S. W. Cho, J. Cardinal, T. R. Billiar, D. A. Geller, A. Tsung; University of Pittsburgh Medical Center, Pittsburgh, PA
46.8. Single Cell Analysis Demonstrates Significant Transcriptional Heterogeneity Within Tightly-Sorted Murine Hematopoietic Stem Cell Populations. J. P. Glotzbach, I. N. Vial, M. Januszyk, V. W. Wong, H. Thangarajah, M. T. Longaker, G. C. Gurtner; Stanford University, Stanford, CA
Background: Danger associated molecular patterns (DAMPs) initiate inflammatory pathways that are common to both sterile and infectious processes. One such DAMP, high molecular group box-1 (HMGB-1), is a nuclear protein that is released from stressed hepatocytes and mediates inflammation after liver ischemia/reperfusion (I/R) injury. We sought to determine the role of the transcription factor, Interferon Regulatory Factor-1 (IRF-1), in the mechanism of HMGB-1 release following ischemic liver injury. Methods: For in