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Silencing VEGF by short interfering RNA chemical transfection decreases mesenchymal stem cell mediated cardioprotection
CARDIOTHORACIC SURGERY I RESULTS: One hour following complex cardiac surgery, the quartile of patients with the greatest IL-6 response also had the greatest increase in serum NGAL (145%) compared to the lowest IL-6 quartile (68%, p⫽0.05), and 70% of these patients progressed on to clinical kidney injury. Furthermore, six hours following surgery, the quartile of patients with the greatest IL-10 response showed a larger increase in NGAL from baseline compared to the lowest IL-10 quartile (104% vs. 19% p⫽0.01), and these patients also had more pulmonary failure (60% vs 10%, p⫽0.01) and multiple organ failure (70% vs 30%, p⫽0.02) with longer ICU and hospital stays (p⫽0.001) (Table).
Hemodynamics of transcatheter aortic valve stenosis Harry Dwyer PhD, Peter B Matthews BS, Ali Azadani PhD, Liang Ge PhD, David Saloner PhD, T Sloane Guy MD, Elaine E Tseng MD University of California at Davis, Davis, CA INTRODUCTION: Transcatheter aortic valves (TAV) present a minimally invasive treatment for high risk surgical patients with severe aortic stenosis. However, their long-term safety and functionality are unknown. The objective of this study is to evaluate hemodynamic changes within TAV created by bioprosthetic leaflet degeneration. METHODS: Three computational fluid dynamics simulations were performed to evaluate the hemodynamics through normal, mildly and severely stenosed TAV. A surface mesh of the aortic root with the TAV attached at the annulus was generated. Unsteady flow analysis (320 computational steps per second) throughout systole was used to calculate fluid velocity, and shear and total force on the mesh elements.
6 Hour Serum Cytokine Quartiles for IL-6 and IL-10
RESULTS: Mild stenosis increased total force on the TAV by over 60% (0.602 to 0.98 N), severe stenosis another 80% (1.79 N). Of the calculated force, 99% was in the direction of axial flow, along the aortic surface of the prosthetic leaflets. Mild stenosis increased average but not maximum shear stress on the leaflet and sinus surfaces. Severe stenosis led to a dramatic increase in maximum shear stress, occurring at the leaflet tips during peak flow (120 KPa severe v. 45 KPa normal/mild).
IL-6
IL-6
IL-6
IL-10
IL-10
IL-10
Lowest Quartile Median (121 pg/ ml)
Highest Quartile Median (486 pg/ ml)
p-value
Lowest Quartile Median (10 pg/ ml)
Highest Quartile Median (289 pg/ ml)
p-value
Pulmonary Failure, n
0
5
0.009
1
6
0.01
Time to Extubation, median (hrs)
18
32
NS
16
45
0.004
Multi-Organ Failure, n
2
4
NS
3
7
0.04
NGAL ( % change from baseline)
33
78
0.17
19
104
0.01
ICU stay, median days
3
6
0.03
4
7
0.03
Total length of stay, median days
8
17
0.06
7
22
0.001
CONCLUSIONS: NGAL and inflammatory biomarkers were elevated hours following surgery in those patients that developed increased kidney and pulmonary failure. Timely identification of patients at risk for organ injury may allow for early intervention and reduce resource utilization.
CONCLUSIONS: Stenosis leads to significant forces on TAV during systole. Shear stress has long been recognized as a mechanism of leaflet degeneration and can lead to stent migration over time. As the first TAV begin to stenose, the authors recommend watchful examination for device failure.
Early blood biomarkers predict organ injury and resource utilization following complex cardiac surgery
Silencing VEGF by short interfering RNA chemical transfection decreases mesenchymal stem cell mediated cardioprotection
Tad Kim MD, George J Arnaoutakis MD, Azra Bihorac MD, Tomas D Martin MD, Phillip J Hess Jr MD, Charles T Klodell MD, Curtis G Tribble MD, Ahsan A Ejaz MD, Lyle L Moldawer PhD, Thomas M Beaver MD, MPH University of Florida College of Medicine, Gainesville, FL
Paul R Crisostomo MD, Yue Wang PhD, Troy A Markel MD, Nathan M Novotny MD, Rinki Ray MD, Daniel R Meldrum MD, FACS Indiana University, Indianapolis, IN INTRODUCTION: Exogenous mesenchymal stem cell(MSC) transplantation is becoming an increasingly realistic treatment modality and may repair injured tissue via the release of cytoprotective factors. VEGF is a central growth and survival factor for both the endothelium and the myocardium. However, it remains unknown whether MSC paracrine secretion of VEGF is a critical component of MSC mediated cardioprotection. We hypothesize that siRNA transfection targeting the VEGF-A gene in MSCs will decrease MSC and conditioned media derived cardioprotection during myocardial ischemia.
INTRODUCTION: Patients undergoing complex cardiac surgery are at risk for organ failure. Neutrophil-gelatinase-associatedlipocalin (NGAL) has been found to be an early biomarker for renal injury. Multiplex immunoassays facilitate evaluation of the early inflammatory response. We examined biomarker appearance (NGAL and multiplex cytokines) with organ injury and resource utilization. METHODS: NGAL and multiplex cytokine immunoassays were performed at baseline, 1, 6, and 24 hours following surgery on 38 patients undergoing thoracic aorta and valve operations. Mean age was 65 yrs with 26 males and 12 females. Acute kidney injury (AKIN definition), pulmonary failure (⬎48 hrs intubation), multiple-organ failure (MOF), and intensive care unit and hospital stays were measured.
© 2008 by the American College of Surgeons Published by Elsevier Inc.
METHODS: Male murine MSCs were harvested, cultured, and divided into groups: 1)untreated control; 2)MSCs transfected with scramble control; 3)MSCs transfected with siRNA targeting the
ISSN 1072-7515/08/$34.00
S26
Vol. 207, No. 3S, September 2008
VEGF-A gene(100nM). Adult male SD rat hearts (n⫽20) were isolated, perfused (Langendorff ), and subjected to 15 min equilibration, 25 min warm global ischemia, and 40 min reperfusion. Experimental hearts were infused with 1x106 MSCs or cell-free conditioned media prior to ischemia. Data reported as mean⫾SEM, p⬍0.05, ANOVA and Tukeys. RESULTS: Post-ischemic recovery of left ventricular developed pressure (%equilibration) was significantly greater with MSCs (46.2⫾4.1%) and MSC conditioned media (44.2⫾4.0%) than with vehicle (35.3⫾2.7%) at end reperfusion. However, hearts treated with siRNA VEGF deleted MSCs (29.7⫾1.7%) and corresponding conditioned media (28.5⫾5.4%) exhibited no difference from vehicle. No significant difference was observed between hearts treated with MSCs or siRNA scramble control MSCs. End diastolic pressure and ⫾dP/dt followed a similar pattern in post ischemic functional recovery. CONCLUSIONS: Targeted deletion of MSC VEGF by siRNA transfection abolishes acute MSC and MSC derived conditioned media cardioprotection. These findings may help elucidate the critical components of MSC paracrine repair.
Ventricular restraint therapy: And what of the right ventricle? Ravi Kiran Ghanta MD, Lawrence Lee MD, Arvind T Rangaraj MD, Ramanan Umakanthan MD, Rita G Laurence BS, R Morton Bolman MD, Lawrence H Cohn MD, Frederick Y Chen MD, PhD Brigham and Women’s Hospital, Boston, MA INTRODUCTION: Previous studies have shown that ventricular restraint decreases left ventricular (LV) transmural pressure (Ptm) and promotes reverse remodeling in heart failure. No study has evaluated the effect of restraint on the right ventricle (RV). In this study, the effects of wrap tightness on RV and LV Ptm were compared. METHODS: A fluid-filled epicardial balloon previously developed was implanted around the ventricles of 5 adult ovine. Restraint levels were defined by the maximum pressure applied by the balloon to the epicardium. This occurs at end-diastole. In each ovine, we simultaneously measured aortic, LV, RV, and epicardial pressure throughout the cardiac cycle at 5 increasing restraint levels: 0, 1.5, 3.0, 5.0, and 8.0 mmHg. Ptm was defined as ventricular pressure minus epicardial pressure. Transeptal pressure (Pseptum) was defined as LV minus RV pressure. Repeated measures analysis of variance was then used to evaluate change in LV Ptm, RV Ptm, and Pseptum from baseline. RESULTS: Ventricular restraint significantly decreased LV Ptm but had no effect on RV Ptm. As restraint level was increased, diastolic RV pressure increased correspondingly. The increase in RV enddiastolic pressure (EDP) correlated with the increase in wrap tightness (r2⫽0.96). This increase in RV pressure led to a decrease in Pseptum.
Surgical Forum Abstracts
LV Restraint Level (mmHg)
RV
S27
Septum
Mean LV Ptm (mmHg)
Mean RV Ptm (mmHg)
RV EDP (mmHg)
⌬ RV EDP (mmHg)
Mean Pseptum (mmHg)
0
37.2
8.4
2.8
0.0
28.8
1.5
35.2
8.7
4.2
1.3
26.5
3.0
30.9ⴱ
9.6
6.2ⴱ
3.4
21.3ⴱ
5.0
26.9ⴱ
10.4
8.1ⴱ
5.3
16.6ⴱ
8.0
19.4ⴱ
10.6
11.5ⴱ
8.7
11.1ⴱ
ⴱp⬍0.05 change from baseline.
CONCLUSIONS: Unlike the LV, ventricular restraint therapy does not alter RV Ptm. Epicardial pressure applied to the compliant RV is instead transmitted to the septum. Measurement of RV EDP may be used to estimate wrap tightness with ventricular restraint and possibly guide placement.
Profound hypothermia decreases cardiac apoptosis through preservation of Akt survival pathway Fahad Shuja MD, Malek Tabbara MD, Yongqing Li MD, PhD, Baoling Liu MD, Muhammad U Butt MD, Elizabeth A Sailhamer MD, George C Velmahos MD, FACS, Marc A deMoya MD, Hasan B Alam MD, FACS Massachusetts General Hospital/Harvard Medical School, Boston, MA INTRODUCTION: We have previously demonstrated that induction of profound hypothermia improves long term survival in large animal models of complex injuries/lethal hemorrhage. However, the precise mechanisms were not clear. This study was done to investigate the effects of profound hypothermia on the Akt cell survival pathway in a rodent model of hemorrhage. METHODS: Sprague-Dawley rats were subjected to 40% hemorrhage over 10 minutes, and cardiopulmonary bypass (CPB) was used to modulate the core temp over 30 minutes (n⫽3/group) as follows: 1) Profound hypothermic shock (HS; core temp 15°C), and 2) Normothermic shock (NS; temp 36-37°C). Target temperature was maintained using low flow CPB during 60 minutes of shock. Animals were then resuscitated (and re-warmed) on full CPB over 40 minutes, and monitored for 3 hours. Sham rats (no hemorrhage/ resuscitation) served as controls. Blood samples were collected serially and cardiac tissues were harvested at end experiment. Levels of Akt and its downstream targets were measured using Western Blot. RESULTS: Survival rates in HS and NS groups were 100% and 0% respectively. Severity of lactic acidosis was significantly higher after NS (p⫽0.002 vs. HS). Phosphorylated-Akt (active) was increased in HS and Sham groups compared to NS (p⫽0.05). Among downstream targets of P-Akt, bcl-2 (anti-apoptotic) was increased while activated caspase-3 (pro-apoptotic) was decreased in HS (p⬍0.001 vs. NS). The protein profile in HS animals was identical to the Sham. CONCLUSIONS: Induction of profound hypothermia increases survival in a rodent model of hemorrhagic shock, in part through preservation of the Akt cell survival pathway.
Hemodynamics of transcatheter aortic valve stenosis Harry Dwyer PhD, Peter B Matthews BS, Ali Azadani PhD, Liang Ge PhD, David Saloner PhD, T Sloane Guy MD, Elaine E Tseng MD University of California at Davis, Davis, CA INTRODUCTION: Transcatheter aortic valves (TAV) present a minimally invasive treatment for high risk surgical patients with severe aortic stenosis. However, their long-term safety and functionality are unknown. The objective of this study is to evaluate hemodynamic changes within TAV created by bioprosthetic leaflet degeneration. METHODS: Three computational fluid dynamics simulations were performed to evaluate the hemodynamics through normal, mildly and severely stenosed TAV. A surface mesh of the aortic root with the TAV attached at the annulus was generated. Unsteady flow analysis (320 computational steps per second) throughout systole was used to calculate fluid velocity, and shear and total force on the mesh elements.
6 Hour Serum Cytokine Quartiles for IL-6 and IL-10
RESULTS: Mild stenosis increased total force on the TAV by over 60% (0.602 to 0.98 N), severe stenosis another 80% (1.79 N). Of the calculated force, 99% was in the direction of axial flow, along the aortic surface of the prosthetic leaflets. Mild stenosis increased average but not maximum shear stress on the leaflet and sinus surfaces. Severe stenosis led to a dramatic increase in maximum shear stress, occurring at the leaflet tips during peak flow (120 KPa severe v. 45 KPa normal/mild).
IL-6
IL-6
IL-6
IL-10
IL-10
IL-10
Lowest Quartile Median (121 pg/ ml)
Highest Quartile Median (486 pg/ ml)
p-value
Lowest Quartile Median (10 pg/ ml)
Highest Quartile Median (289 pg/ ml)
p-value
Pulmonary Failure, n
0
5
0.009
1
6
0.01
Time to Extubation, median (hrs)
18
32
NS
16
45
0.004
Multi-Organ Failure, n
2
4
NS
3
7
0.04
NGAL ( % change from baseline)
33
78
0.17
19
104
0.01
ICU stay, median days
3
6
0.03
4
7
0.03
Total length of stay, median days
8
17
0.06
7
22
0.001
CONCLUSIONS: NGAL and inflammatory biomarkers were elevated hours following surgery in those patients that developed increased kidney and pulmonary failure. Timely identification of patients at risk for organ injury may allow for early intervention and reduce resource utilization.
CONCLUSIONS: Stenosis leads to significant forces on TAV during systole. Shear stress has long been recognized as a mechanism of leaflet degeneration and can lead to stent migration over time. As the first TAV begin to stenose, the authors recommend watchful examination for device failure.
Early blood biomarkers predict organ injury and resource utilization following complex cardiac surgery
Silencing VEGF by short interfering RNA chemical transfection decreases mesenchymal stem cell mediated cardioprotection
Tad Kim MD, George J Arnaoutakis MD, Azra Bihorac MD, Tomas D Martin MD, Phillip J Hess Jr MD, Charles T Klodell MD, Curtis G Tribble MD, Ahsan A Ejaz MD, Lyle L Moldawer PhD, Thomas M Beaver MD, MPH University of Florida College of Medicine, Gainesville, FL
Paul R Crisostomo MD, Yue Wang PhD, Troy A Markel MD, Nathan M Novotny MD, Rinki Ray MD, Daniel R Meldrum MD, FACS Indiana University, Indianapolis, IN INTRODUCTION: Exogenous mesenchymal stem cell(MSC) transplantation is becoming an increasingly realistic treatment modality and may repair injured tissue via the release of cytoprotective factors. VEGF is a central growth and survival factor for both the endothelium and the myocardium. However, it remains unknown whether MSC paracrine secretion of VEGF is a critical component of MSC mediated cardioprotection. We hypothesize that siRNA transfection targeting the VEGF-A gene in MSCs will decrease MSC and conditioned media derived cardioprotection during myocardial ischemia.
INTRODUCTION: Patients undergoing complex cardiac surgery are at risk for organ failure. Neutrophil-gelatinase-associatedlipocalin (NGAL) has been found to be an early biomarker for renal injury. Multiplex immunoassays facilitate evaluation of the early inflammatory response. We examined biomarker appearance (NGAL and multiplex cytokines) with organ injury and resource utilization. METHODS: NGAL and multiplex cytokine immunoassays were performed at baseline, 1, 6, and 24 hours following surgery on 38 patients undergoing thoracic aorta and valve operations. Mean age was 65 yrs with 26 males and 12 females. Acute kidney injury (AKIN definition), pulmonary failure (⬎48 hrs intubation), multiple-organ failure (MOF), and intensive care unit and hospital stays were measured.
© 2008 by the American College of Surgeons Published by Elsevier Inc.
METHODS: Male murine MSCs were harvested, cultured, and divided into groups: 1)untreated control; 2)MSCs transfected with scramble control; 3)MSCs transfected with siRNA targeting the
ISSN 1072-7515/08/$34.00
S26
Vol. 207, No. 3S, September 2008
VEGF-A gene(100nM). Adult male SD rat hearts (n⫽20) were isolated, perfused (Langendorff ), and subjected to 15 min equilibration, 25 min warm global ischemia, and 40 min reperfusion. Experimental hearts were infused with 1x106 MSCs or cell-free conditioned media prior to ischemia. Data reported as mean⫾SEM, p⬍0.05, ANOVA and Tukeys. RESULTS: Post-ischemic recovery of left ventricular developed pressure (%equilibration) was significantly greater with MSCs (46.2⫾4.1%) and MSC conditioned media (44.2⫾4.0%) than with vehicle (35.3⫾2.7%) at end reperfusion. However, hearts treated with siRNA VEGF deleted MSCs (29.7⫾1.7%) and corresponding conditioned media (28.5⫾5.4%) exhibited no difference from vehicle. No significant difference was observed between hearts treated with MSCs or siRNA scramble control MSCs. End diastolic pressure and ⫾dP/dt followed a similar pattern in post ischemic functional recovery. CONCLUSIONS: Targeted deletion of MSC VEGF by siRNA transfection abolishes acute MSC and MSC derived conditioned media cardioprotection. These findings may help elucidate the critical components of MSC paracrine repair.
Ventricular restraint therapy: And what of the right ventricle? Ravi Kiran Ghanta MD, Lawrence Lee MD, Arvind T Rangaraj MD, Ramanan Umakanthan MD, Rita G Laurence BS, R Morton Bolman MD, Lawrence H Cohn MD, Frederick Y Chen MD, PhD Brigham and Women’s Hospital, Boston, MA INTRODUCTION: Previous studies have shown that ventricular restraint decreases left ventricular (LV) transmural pressure (Ptm) and promotes reverse remodeling in heart failure. No study has evaluated the effect of restraint on the right ventricle (RV). In this study, the effects of wrap tightness on RV and LV Ptm were compared. METHODS: A fluid-filled epicardial balloon previously developed was implanted around the ventricles of 5 adult ovine. Restraint levels were defined by the maximum pressure applied by the balloon to the epicardium. This occurs at end-diastole. In each ovine, we simultaneously measured aortic, LV, RV, and epicardial pressure throughout the cardiac cycle at 5 increasing restraint levels: 0, 1.5, 3.0, 5.0, and 8.0 mmHg. Ptm was defined as ventricular pressure minus epicardial pressure. Transeptal pressure (Pseptum) was defined as LV minus RV pressure. Repeated measures analysis of variance was then used to evaluate change in LV Ptm, RV Ptm, and Pseptum from baseline. RESULTS: Ventricular restraint significantly decreased LV Ptm but had no effect on RV Ptm. As restraint level was increased, diastolic RV pressure increased correspondingly. The increase in RV enddiastolic pressure (EDP) correlated with the increase in wrap tightness (r2⫽0.96). This increase in RV pressure led to a decrease in Pseptum.
Surgical Forum Abstracts
LV Restraint Level (mmHg)
RV
S27
Septum
Mean LV Ptm (mmHg)
Mean RV Ptm (mmHg)
RV EDP (mmHg)
⌬ RV EDP (mmHg)
Mean Pseptum (mmHg)
0
37.2
8.4
2.8
0.0
28.8
1.5
35.2
8.7
4.2
1.3
26.5
3.0
30.9ⴱ
9.6
6.2ⴱ
3.4
21.3ⴱ
5.0
26.9ⴱ
10.4
8.1ⴱ
5.3
16.6ⴱ
8.0
19.4ⴱ
10.6
11.5ⴱ
8.7
11.1ⴱ
ⴱp⬍0.05 change from baseline.
CONCLUSIONS: Unlike the LV, ventricular restraint therapy does not alter RV Ptm. Epicardial pressure applied to the compliant RV is instead transmitted to the septum. Measurement of RV EDP may be used to estimate wrap tightness with ventricular restraint and possibly guide placement.
Profound hypothermia decreases cardiac apoptosis through preservation of Akt survival pathway Fahad Shuja MD, Malek Tabbara MD, Yongqing Li MD, PhD, Baoling Liu MD, Muhammad U Butt MD, Elizabeth A Sailhamer MD, George C Velmahos MD, FACS, Marc A deMoya MD, Hasan B Alam MD, FACS Massachusetts General Hospital/Harvard Medical School, Boston, MA INTRODUCTION: We have previously demonstrated that induction of profound hypothermia improves long term survival in large animal models of complex injuries/lethal hemorrhage. However, the precise mechanisms were not clear. This study was done to investigate the effects of profound hypothermia on the Akt cell survival pathway in a rodent model of hemorrhage. METHODS: Sprague-Dawley rats were subjected to 40% hemorrhage over 10 minutes, and cardiopulmonary bypass (CPB) was used to modulate the core temp over 30 minutes (n⫽3/group) as follows: 1) Profound hypothermic shock (HS; core temp 15°C), and 2) Normothermic shock (NS; temp 36-37°C). Target temperature was maintained using low flow CPB during 60 minutes of shock. Animals were then resuscitated (and re-warmed) on full CPB over 40 minutes, and monitored for 3 hours. Sham rats (no hemorrhage/ resuscitation) served as controls. Blood samples were collected serially and cardiac tissues were harvested at end experiment. Levels of Akt and its downstream targets were measured using Western Blot. RESULTS: Survival rates in HS and NS groups were 100% and 0% respectively. Severity of lactic acidosis was significantly higher after NS (p⫽0.002 vs. HS). Phosphorylated-Akt (active) was increased in HS and Sham groups compared to NS (p⫽0.05). Among downstream targets of P-Akt, bcl-2 (anti-apoptotic) was increased while activated caspase-3 (pro-apoptotic) was decreased in HS (p⬍0.001 vs. NS). The protein profile in HS animals was identical to the Sham. CONCLUSIONS: Induction of profound hypothermia increases survival in a rodent model of hemorrhagic shock, in part through preservation of the Akt cell survival pathway.