GAL4CNFkappaB Fusion Protein Augments Transgene Expression ...
Sodium butyrate markedly augments pro-apoptotic gene therapy via alterations in bcl-2 family protein...
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rected against phosphorylated ERK1/2. Apoptosis was measured as a percentage of DNA strand breaks by TUNEL method. Results: SNAP reduced levels of ap- optosis to below control levels (Fig. 1a). The addition of ODQ to SNAP- treated cells increased levels to above control levels, which then fell with addition of 8-bromo-cGMP (Fig- ure 1a). SNAP increased levels of phosphorylated ERK1/2, an effect eliminated by ODQ and restored with cGMP (Figure1b). Conclusions: NO inhibition of apo- ptosis is dependent on cGMP, an effect associated with ERK1/2 activation. Cardiac gene transfer of HGF is superior to TMR in inducing therapeutic coronary neoangiogenesis AS Stewart, MD, LT Bish, DR Brinster, HL Sweeney, Ph.D, TJ Gardner, MD, FACS. University of Pennsylvania School of Medicine Department of Surgery, University of Pennsylvania School of Medicine, 4 Silverstein Pavilion, 3400 Spruce Street, Philadelphia, PA 19104. USA Introduction: Transmyocardial Laser Revascularization (TMLR) is be- lieved to improve perfusion to ischemic myocardium through the cre- ation of multiple areas of inflammation. Recently, the benefits of TMLR were reproduced with Transmyocardial Mechanical Revascularization: the creation of multiple needle punctures into ischemic myocardium. Gene transfer of Hepatocyte Growth Factor (HGF), however, induces neoangiogenesis by stimulating endothelial cell mitogenesis and mo- togenesis. This study quantitatively compares the two methods in terms of vessel formation, infarct protection, and restoration of contractility. Methods: Thirty rabbits underwent left thoracotomy with placement of an ameroid constrictor in the left circumflex artery. Fourteen days after operation, the animals were returned to surgery and randomized to receive TMMR or Adeno-HGF, or no intervention. TMMR animals received 20 punctures with a 19g needle. The HGF group received adeno-HGF, in concert with 2mg of Hyalurondase and 2mg Collage- nase, as a single apical injection. Four weeks post-intervention, the animals were returned for non-survival surgery. Two, one mm micro- crystals were placed in the area of injection to assess contractility. The hearts were then procured, sectioned, and stained for endothelial cell proliferation and microsphere quantification. Results: Significant increases in small and medium size arterioles were demonstrated in the HGF group, compared to the TMMR and control population. A significant reduction in the percent infarction of the circumflex distribution was demonstrated in the HGF group, as well as a reduction of the apoptotic index in the area at risk. Finally, the injection of adeno-HGF restores a significantly higher percentage of baseline contractility, compared to TMMR treated hearts. Conclusions: The data support the superiority of adenoviral gene transfer of HGF in inducing therapeutic coronary neoangiogenesis. Administration of adeno-HGF results in a significant increase in coro- nary neoangiogenesis, a dimunition of infarct size, a reduction in apoptosis, and a restoration of myocardial contractility in the circum- flex distribution, compared to TMMR. Sodium butyrate markedly augments pro-apoptotic gene therapy via alterations in bcl-2 family protein expression Imran Mohiuddin, M.D., Xiaobo Cao, M.D., W. Roy Smythe, M.D. Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Box 109, 1515 Holcombe Blvd., Houston, TX 77030, USA Phone: 713/792-6933; Fax: 713/794- 4901 Introduction: The ratio of pro-apoptotic (PAP) and anti-apoptotic (AAP) bcl-2 family proteins is important in regulation of apoptosis. Recombinant adenovirus capable of transducing cells with PAP (PAV) have been developed (Adbak, Adbax). Sodium butyrate (NB) is a dif- ferentiating agent reportedly capable of downregulation of AAP. We sought to determine if therapy with Adbak and Adbax with NB would augment apoptosis and cell killing by a combination of up-regulation of PAP and down-regulation of AAP. Methods: Human mesothelioma (REN, I-45) and non small cell lung carcinoma (A549, H1299) cell lines were utilized. Cells were exposed to Adbak, Adbax and NB alone as well as in combination (PAV/NB) at varying doses and time points. Cellular death was assessed by a colorimetric (XTT) assay. Apoptosis was evaluated by Hoescht stain and quantified by FACS subG1 analysis. Isobolograms were con- structed to evaluate additive or synergistic effect. Results: Cellular death was augmented in all lines by PAV/NB. Apo- ptosis was highest in the PAV/NB groups with an increase in apoptotic cell population by an average of 54 percent compared to single agent alone. Following PAV/NB therapy, a decrease of the AAP bcl-2 was noted in combination with increases in PAP bak (adbak) or bax (adbax). By isobologram analysis, PAV/NB treatment mediated cell killing proved to be synergistic in all lines. Conclusions: PAV/NB proved more effective than any single agent in induction of apoptosis, and cellular killing was synergistic. Synergy may be due to the ability of NB to decrease AAP levels with marked increases in PAP engendered by transfer of bak and bax. Combination therapy with differentiating agents such as NB in addition to PAV may prove useful clinically. Neurological Surgery Thrombin reduces cerebral artery contractions caused by cerebrospinal fluid from subarachnoid hemorrhage patients Mark K Borsody, M.D., Ph.D., Gina M DeGiovanni, B.S., Linda S Marton, Ph.D., R. Loch Macdonald, M.D., Ph.D., Bryce Weir, M.D. Section of Neurosurgery, The University of Chicago, 5841 S. Maryland Av., Chicago, IL 60637, USA TEL: (773) 702-2123 Introduction: We observed that the second application of cerebrospi- nal fluid (CSF) from subarachnoid hemorrhage (SAH) patients onto cerebral arteries ex vivo produces a greater contraction than does the initial application. We hypothesized that the initial reduction of SAH CSF vasoconstriction is caused by the proteolytic activity of thrombin, and that thrombin also reduces the tension generated by the chief vasoconstrictor in SAH CSF, hemoglobin (Hb). Methods: The vertebrobasilar arteries were removed from dogs under general anesthesia, cut into 4 mm rings and suspended in tissue culture baths to measure isometric tension generated by the seg- ments. CSF was taken from patients 3–7 days post-SAH via ventricular drains. CSF was administered in 10 25 2 10 21 dilutions. The thrombin antagonist hirudin (5 U) was administered before CSF in some exper- iments. The arterial tension responses to pure Hb (10 24 2 3.2 g/dl) and thrombin (10 24 2 3.2 U/ml), administered alone or in combination, were also examined. Results: The initial application of SAH CSF increased artery segment tension by 15 6 2% at the maximum CSF concentration. Following an extensive washout, the second application of those CSF samples to the same artery segments increased tension 30 6 9%. Hirudin aug- mented the tension generated by the initial SAH CSF application, but had no effect on the tension generated by the second CSF application. While examining this phenomenon, we found that thrombin or Hb administered alone caused only vasoconstriction. However, thrombin administered together with Hb reduced the tension generated by Hb, rather than increasing it in an additive fashion. Conclusions: Thrombin in SAH CSF acts to limit the contraction of cerebral arteries ex vivo caused by the CSF. This may involve antag- onism of Hb-induced vasoconstriction by thrombin. S38 Surgical Forum Abstracts J Am Coll Surg
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Transcript of Sodium butyrate markedly augments pro-apoptotic gene therapy via alterations in bcl-2 family protein...
rected against phosphorylatedERK1/2. Apoptosis was measured asa percentage of DNA strand breaksby TUNEL method.
Results: SNAP reduced levels of ap-optosis to below control levels (Fig.1a). The addition of ODQ to SNAP-treated cells increased levels toabove control levels, which then fellwith addition of 8-bromo-cGMP (Fig-ure 1a). SNAP increased levels ofphosphorylated ERK1/2, an effecteliminated by ODQ and restored withcGMP (Figure1b).
Conclusions: NO inhibition of apo-ptosis is dependent on cGMP, an effect associated with ERK1/2activation.
Cardiac gene transfer of HGF is superior to TMR ininducing therapeutic coronary neoangiogenesisAS Stewart, MD, LT Bish, DR Brinster, HL Sweeney, Ph.D, TJGardner, MD, FACS. University of Pennsylvania School of MedicineDepartment of Surgery, University of Pennsylvania School ofMedicine, 4 Silverstein Pavilion, 3400 Spruce Street, Philadelphia,PA 19104. USA
Introduction: Transmyocardial Laser Revascularization (TMLR) is be-lieved to improve perfusion to ischemic myocardium through the cre-ation of multiple areas of inflammation. Recently, the benefits of TMLRwere reproduced with Transmyocardial Mechanical Revascularization:the creation of multiple needle punctures into ischemic myocardium.Gene transfer of Hepatocyte Growth Factor (HGF), however, inducesneoangiogenesis by stimulating endothelial cell mitogenesis and mo-togenesis. This study quantitatively compares the two methods interms of vessel formation, infarct protection, and restoration ofcontractility.
Methods: Thirty rabbits underwent left thoracotomy with placement ofan ameroid constrictor in the left circumflex artery. Fourteen days afteroperation, the animals were returned to surgery and randomized toreceive TMMR or Adeno-HGF, or no intervention. TMMR animalsreceived 20 punctures with a 19g needle. The HGF group receivedadeno-HGF, in concert with 2mg of Hyalurondase and 2mg Collage-nase, as a single apical injection. Four weeks post-intervention, theanimals were returned for non-survival surgery. Two, one mm micro-crystals were placed in the area of injection to assess contractility. Thehearts were then procured, sectioned, and stained for endothelial cellproliferation and microsphere quantification.
Results: Significant increases in small and medium size arterioles weredemonstrated in the HGF group, compared to the TMMR and controlpopulation. A significant reduction in the percent infarction of thecircumflex distribution was demonstrated in the HGF group, as well asa reduction of the apoptotic index in the area at risk. Finally, theinjection of adeno-HGF restores a significantly higher percentage ofbaseline contractility, compared to TMMR treated hearts.
Conclusions: The data support the superiority of adenoviral genetransfer of HGF in inducing therapeutic coronary neoangiogenesis.Administration of adeno-HGF results in a significant increase in coro-nary neoangiogenesis, a dimunition of infarct size, a reduction inapoptosis, and a restoration of myocardial contractility in the circum-flex distribution, compared to TMMR.
Sodium butyrate markedly augments pro-apoptoticgene therapy via alterations in bcl-2 family proteinexpressionImran Mohiuddin, M.D., Xiaobo Cao, M.D., W. Roy Smythe, M.D.Department of Thoracic and Cardiovascular Surgery, The Universityof Texas M.D. Anderson Cancer Center, Box 109, 1515 HolcombeBlvd., Houston, TX 77030, USA Phone: 713/792-6933; Fax: 713/794-4901
Introduction: The ratio of pro-apoptotic (PAP) and anti-apoptotic(AAP) bcl-2 family proteins is important in regulation of apoptosis.Recombinant adenovirus capable of transducing cells with PAP (PAV)have been developed (Adbak, Adbax). Sodium butyrate (NB) is a dif-ferentiating agent reportedly capable of downregulation of AAP. Wesought to determine if therapy with Adbak and Adbax with NB wouldaugment apoptosis and cell killing by a combination of up-regulation ofPAP and down-regulation of AAP.
Methods: Human mesothelioma (REN, I-45) and non small cell lungcarcinoma (A549, H1299) cell lines were utilized. Cells were exposed toAdbak, Adbax and NB alone as well as in combination (PAV/NB) atvarying doses and time points. Cellular death was assessed by acolorimetric (XTT) assay. Apoptosis was evaluated by Hoescht stainand quantified by FACS subG1 analysis. Isobolograms were con-structed to evaluate additive or synergistic effect.
Results: Cellular death was augmented in all lines by PAV/NB. Apo-ptosis was highest in the PAV/NB groups with an increase in apoptoticcell population by an average of 54 percent compared to single agentalone. Following PAV/NB therapy, a decrease of the AAP bcl-2 wasnoted in combination with increases in PAP bak (adbak) or bax (adbax).By isobologram analysis, PAV/NB treatment mediated cell killingproved to be synergistic in all lines.
Conclusions: PAV/NB proved more effective than any single agent ininduction of apoptosis, and cellular killing was synergistic. Synergymay be due to the ability of NB to decrease AAP levels with markedincreases in PAP engendered by transfer of bak and bax. Combinationtherapy with differentiating agents such as NB in addition to PAV mayprove useful clinically.
Neurological SurgeryThrombin reduces cerebral artery contractionscaused by cerebrospinal fluid from subarachnoidhemorrhage patientsMark K Borsody, M.D., Ph.D., Gina M DeGiovanni, B.S., Linda SMarton, Ph.D., R. Loch Macdonald, M.D., Ph.D., Bryce Weir, M.D.Section of Neurosurgery, The University of Chicago, 5841 S.Maryland Av., Chicago, IL 60637, USA TEL: (773) 702-2123
Introduction: We observed that the second application of cerebrospi-nal fluid (CSF) from subarachnoid hemorrhage (SAH) patients ontocerebral arteries ex vivo produces a greater contraction than does theinitial application. We hypothesized that the initial reduction of SAHCSF vasoconstriction is caused by the proteolytic activity of thrombin,and that thrombin also reduces the tension generated by the chiefvasoconstrictor in SAH CSF, hemoglobin (Hb).
Methods: The vertebrobasilar arteries were removed from dogs undergeneral anesthesia, cut into 4 mm rings and suspended in tissueculture baths to measure isometric tension generated by the seg-ments. CSF was taken from patients 3–7 days post-SAH via ventriculardrains. CSF was administered in 1025 2 1021 dilutions. The thrombinantagonist hirudin (5 U) was administered before CSF in some exper-iments. The arterial tension responses to pure Hb (1024 2 3.2 g/dl) andthrombin (1024 2 3.2 U/ml), administered alone or in combination,were also examined.
Results: The initial application of SAH CSF increased artery segmenttension by 15 6 2% at the maximum CSF concentration. Following anextensive washout, the second application of those CSF samples tothe same artery segments increased tension 30 6 9%. Hirudin aug-mented the tension generated by the initial SAH CSF application, buthad no effect on the tension generated by the second CSF application.While examining this phenomenon, we found that thrombin or Hbadministered alone caused only vasoconstriction. However, thrombinadministered together with Hb reduced the tension generated by Hb,rather than increasing it in an additive fashion.
Conclusions: Thrombin in SAH CSF acts to limit the contraction ofcerebral arteries ex vivo caused by the CSF. This may involve antag-onism of Hb-induced vasoconstriction by thrombin.
S38 Surgical Forum Abstracts J Am Coll Surg
