- Email: [email protected]
reperfusion
Vol. 211, No. 3S, September 2010
METHODS: HUVEC seeded on glass slides were exposed to T&F or PF in parallel flow chambers in the presence or absence of Thrombin (Th) for 2hr, 4hr and 6hr. TF RNA expression was determined using real-time quantitative RT-PCR and assessed by fold changes compared to that in static control. RESULTS: All groups exposed to mechanical stress and to combination of mechanical and chemical stimuli demonstrated significantly higher TF RNA expressions compared to static control. Comparing effects of mechanical forces, T&F induced significantly higher TF expression than PF, especially at 2hour time point. Furthermore, combination of mechanical and chemical stimuli induced higher TF expression than only mechanical stresses. In addition, T&F in the presence of Th induced significantly higher TF RNA expression than PF and Th, and its induction lasted for 6 hours. 2 hr 4 hr 6 hr Static Control 1.00 ⫾0.0 1.10 ⫾0.1 1.25 ⫾0.2 Pulsatile Forward Flow† 3.14 ⫾0.5 2.83 ⫾0.4 2.93 ⫾0.9 * * To&Fro Flow† 6.63 ⫾0.8 5.58 ⫾1.2 5.41 ⫾2.2 Pulsatile Forward Flow ⫹ 14.90 ⫾1.5 7.44 ⫾1.0 6.21 ⫾1.2 Thrombin† * * * To&Fro Flow ⫹ 18.68 ⫾4.5 15.12 ⫾5.8 15.35 ⫾6.3 Thrombin† Mean ⫾SE, n⫽4-8, †: p⬍0.05, compared to static control by ANOVA and post-hoc analysis of Bonferoni *: p⬍0.05, by ANOVA and post-hoc analysis of Bonferoni.
CONCLUSIONS: Disturbed to&fro flow induced greater and sustained amplification of TF expression in the presence and absence of chemical stimuli. These results may explain increased atherosclerosis in areas of the vascular tree that are exposed to disturbed flow.
Inducible nitric oxide synthase modulates the expression of molecular markers of muscle regeneration following hind limb ischemia reperfusion Bao-Ngoc H Nguyen, MD, Hassan Albadawi MD, Rabih Houbballah MD, Mounir J Haurani MD, Hyung-Jin Yoo MS Massachusetts General Hospital, Boston, MA INTRODUCTION: Inducible nitric oxide synthase (iNOS) expression during sepsis has been implicated in the development of muscle wasting. These experiments were designed to determine whether iNOS expression impedes skeletal muscle regeneration during the regenerative phase of ischemia reperfusion (IR) injury. METHODS: iNOS-null (iNOS⫺/⫺) and C57BL6 (C57) mice were subjected to 3hrs of hindlimb ischemia followed by reperfusion for 1, 7 or 14 days (D1, D7 and D14). Hindlimb function was analyzed using an injury scale; muscle protein poly-ADPribosylation (PAR, marker of PARP enzyme activity due to genomic and inflammatory stress), and myogenin (transcription factor associated with myofibers differentiation) expression were evaluated by immunoblotting. Total and phosphorylated-S473Akt were assessed using ELISA. Percent of injured or regenerating fibers were counted microscopically.
Surgical Forum Abstracts
S139
RESULTS: Both groups had equivalent skeletal muscle fiber injury (C57 87⫾5, iNOS⫺/⫺ 93⫾1, percent p⫽0.3) and functional deficit at D1 IR. Functional recovery was similar at D7 and D14. iNOS⫺/⫺ expressed transiently higher PAR at Day1, then had markedly lower levels at D7 and D14 IR. In contrast iNOS⫺/⫺ expressed higher myogenin levels throughout reperfusion (vs. C57). Muscle fiber morphology, total and phosphorylated-Akt were not different at any time point. Day 1
PAR (AU)
Day 14
iNOSⴚ/ⴚ
C57
iNOSⴚ/ⴚ
C57
4
4
3.5⫾0.2
3.6⫾0.15
2.2⫾0.1
2.1⫾0.2
25⫾ 4
76 ⫾15*
205⫾23
382⫾35*
138⫾35
349⫾12*
Limb Function Score Myogenin (AU)
Day 7
C57
iNOSⴚ/ⴚ
1519⫾183 2358⫾111* 1335⫾163 748⫾176* 843⫾152 484⫾152*
Total-Akt (pg/mg)
20⫾3
26⫾5
113⫾3
112⫾4
96⫾5
96⫾9
p473-Akt (units/mg)
4.1⫾0.3
5.2⫾0.3
28⫾2
31⫾2
27⫾5
23⫾3
*p⬍0.05 compared to C57
CONCLUSIONS: In these settings, iNOS deficiency decreased PARP enzyme activity and increased the level of myogenin in reperfused skeletal muscle. This suggest that iNOS is partially responsible for activation of PARP mediated inflammatory pathways, and suppresses myogenin expression during the regenerative phase of IR. However, iNOS dependent pathways do not appear to impede functional recovery or muscle fiber maturity.
Inhibition of prolyl hydroxylase attenuates microvascular inflammation after mesenteric ischemia/reperfusion Scott M Mullen MD, John G Wood PhD, Michael Moncure MD, James H Thomas MD University of Kansas Medical Center, Kansas City, KS INTRODUCTION: Acute mesenteric ischemia is a life-threatening condition that requires early intervention to improve blood flow. Restoration of blood flow to ischemic bowel results in ischemia/ reperfusion (I/R) injury, and the resulting microvascular inflammation contributes to the high morbidity and mortality associated with this condition. Inhibition of prolyl hydroxylase (PHD) increases hypoxiainducible factor-1 (HIF-1) levels, which promotes expression of genes affecting microvascular function. Our goal was to evaluate whether inhibition of PHD attenuated I/R-induced increases in leukocyte adherence/emigration and vascular permeability (VP), and if this involved upregulation of inducible nitric oxide synthase (iNOS). METHODS: Rats were injected with vehicle or the PHD inhibitor ethyl dihydrobenzoate (EDHB) 24 hr before experiments. Intravital microscopy was used to measure leukocyte adherence/emigration and VP in mesenteric venules of anesthetized rats. Mesentery was superfused with saline or the iNOS inhibitor L-NIL. After a control period, ischemia was produced by placing a microvascular clip on the superior mesenteric artery for 30 min, followed by reperfusion for 2 hrs. RESULTS: Data for leukocyte adherence are presented in the table. Similar results were obtained for leukocyte emigration and VP (data not shown). Leukocyte adherence/emigration and VP increased sig-
S140
Surgical Forum Abstracts
J Am Coll Surg
nificantly after I/R in vehicle-treated rats. However, EDHB significantly attenuated these responses after I/R. L-NIL significantly increased leukocyte adherence/emigration and VP during I/R in EDHB-treated rats. Adherent Leukocytes, # per 100 m venule
Time, min
Control, 15 30 Ischemia, 30 Reperfusion, 15 30 45 60 75 90 105 120
VehicleTreated (n ⴝ 5)
EDHBTreated (n ⴝ 5)
EDHBTreated ⴙ L-NIL (n ⴝ 5)
2.2 ⫾ 0.4 2.0 ⫾ 0.3 -7.0 ⫾ 0.9 7.4 ⫾ 0.5 8.8 ⫾ 0.9 10.2 ⫾ 0.6 10.4 ⫾ 1.1 10.4 ⫾ 1.9 11.0 ⫾ 1.2 12.4 ⫾ 2.9
1.7 ⫾ 0.3 1.7 ⫾ 0.4 -2.0 ⫾ 0.3 1.8 ⫾ 0.4* 2.2 ⫾ 0.4* 2.1 ⫾ 0.3* 2.4 ⫾ 0.4* 2.5 ⫾ 0.3* 2.1 ⫾ 0.3* 2.4 ⫾ 0.4*
1.6 ⫾ 0.3 1.8 ⫾ 0.3 -4.6 ⫾ 0.2 7.0 ⫾ 0.6 8.6 ⫾ 0.5† 8.2 ⫾ 0.4† 8.5 ⫾ 0.9† 8.6 ⫾ 0.6† 9.2 ⫾ 0.7† 9.8 ⫾ 0.8†
Values are means ⫾ standard errors. *indicates p ⬍ 0.05 vs corresponding vehicle-treated animals (ANOVA, specific contrasts). †indicates p ⬍ 0.05 vs corresponding EDHB-treated animals (ANOVA, specific contrasts).
CONCLUSIONS: Inhibition of PHD resulting in upregulation of HIF-1 attenuates microvascular inflammation following mesenteric I/R. This effect involves iNOS upregulation as inhibition of iNOS significantly increased leukocyte adherence/emigration and VP following I/R.
Toll-like receptor 4 mediates oxidized - LDL induced macrophage differentiation to foam cells Kenneth W Howell BS, Xianzhong Meng MD, PhD, David A Fullerton MD, FACS, Chunhua Jin PhD, Lihua Ao BS, T Brett Reece MD, Joseph C Cleveland Jr MD, FACS University of Colorado at Denver School of Medicine, Aurora, CO INTRODUCTION: Macrophage foam cells are central in the development of atherosclerosis but the mechanism of foam cell formation is unclear. Toll-like receptor 4 (TLR4) signaling is known to be important in the pathogenesis of atherosclerosis and oxidized LDL (oxLDL) enhances TLR4 expression in macrophages. We hypothesized that TLR4 is important in macrophage differentiation to foam cells. METHODS: Peritoneal macrophages were isolated by lavage from C3H/HeN (TLR4 competent) and C3H/HeJ (TLR4 dysfunctional) mice. Cells were treated with oxLDL, lipopolysaccharide (LPS) or oxidized LDL plus LPS. Cells were also treated with a TLR4 blocking antibody before oxLDL treatment. Oil red O staining for intracellular lipids was used for identification of foam cells. Statistical analysis was performed using ANOVA with Fisher’s post-test. RESULTS: TLR4 is necessary for macrophage differentiation to foam cells. There is a difference in the percentage of foam cell forma-
tion between TLR4 competent and TLR4 dysfunctional macrophages when treated with oxLDL. Following oxLDL treatment 29% of TLR4 competent cells differentiated to foam cells compared to 5.8% of TLR4 dysfunctional cells (p⬍0.01). Pre-treatment with a TLR4 blocking antibody decreased the differentiation of TLR4 competent cells to foam cells from 29% to 13% (p⬍0.01). Stimulation of TLR4 with LPS in the presence of oxLDL increased differentiation of TLR4 competent cells to foam cells from 29% to 60% (p⬍0.01). CONCLUSIONS: TLR4 is necessary for oxLDL-induced macrophage differentiation to foam cells. We conclude that TLR4 contributes to the pathogenesis of atherosclerosis by promoting foam cell formation.
Endothelial protective and anti-thrombotic effects of proanthocyanidins in a rat model of deep vein thrombosis Yunjian Zhang, Shenming Wang MD, PhD, FACS, Hanping Shi MD, PhD The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China INTRODUCTION: Inflammation plays a crucial role in endothelium injury and the progression of deep vein thrombosis (DVT). The present study was designed to investigate whether proanthocyanidins is a potential therapeutic agent that protects endothelium via the anti-inflammatory property. METHODS: Animal model was performed by the infrarenal inferior vena cava (IVC) ligation to induce DVT. After that, proanthocyanidins (400 mg/kg) was given to rats in the experimental group every 24 h. Vein wall endothelium and thrombi were observed by transmission electron microscopy (TEM). The IVC and iliac/femoral vein were processed for histological analysis. Related inflammatory markers were detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. RESULTS: Proanthocyanidins maintained the integrity of endothelium, reduced the size of thrombus, with up-regulated expression of CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells. Proanthocyanidins decreased the overexpression of von Willebrand factor (vWF) and increased a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) via the anti-inflammatory effects. In addition, the blood levels of inflammatory markers in experimental group were lower than the control (e.g., IL-6, IL-8, TNF-alpha, and LTB4). The expressions of monocyte chemotactic protein-1 (MCP-1), P-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1) were being modulated in experimental group. CONCLUSIONS: In conclusion, our study showed that treatment with proanthocyanidins provides benefit for the vein wall endothelium with the anti-inflammatory property to modulate the progress in thrombosis.
METHODS: HUVEC seeded on glass slides were exposed to T&F or PF in parallel flow chambers in the presence or absence of Thrombin (Th) for 2hr, 4hr and 6hr. TF RNA expression was determined using real-time quantitative RT-PCR and assessed by fold changes compared to that in static control. RESULTS: All groups exposed to mechanical stress and to combination of mechanical and chemical stimuli demonstrated significantly higher TF RNA expressions compared to static control. Comparing effects of mechanical forces, T&F induced significantly higher TF expression than PF, especially at 2hour time point. Furthermore, combination of mechanical and chemical stimuli induced higher TF expression than only mechanical stresses. In addition, T&F in the presence of Th induced significantly higher TF RNA expression than PF and Th, and its induction lasted for 6 hours. 2 hr 4 hr 6 hr Static Control 1.00 ⫾0.0 1.10 ⫾0.1 1.25 ⫾0.2 Pulsatile Forward Flow† 3.14 ⫾0.5 2.83 ⫾0.4 2.93 ⫾0.9 * * To&Fro Flow† 6.63 ⫾0.8 5.58 ⫾1.2 5.41 ⫾2.2 Pulsatile Forward Flow ⫹ 14.90 ⫾1.5 7.44 ⫾1.0 6.21 ⫾1.2 Thrombin† * * * To&Fro Flow ⫹ 18.68 ⫾4.5 15.12 ⫾5.8 15.35 ⫾6.3 Thrombin† Mean ⫾SE, n⫽4-8, †: p⬍0.05, compared to static control by ANOVA and post-hoc analysis of Bonferoni *: p⬍0.05, by ANOVA and post-hoc analysis of Bonferoni.
CONCLUSIONS: Disturbed to&fro flow induced greater and sustained amplification of TF expression in the presence and absence of chemical stimuli. These results may explain increased atherosclerosis in areas of the vascular tree that are exposed to disturbed flow.
Inducible nitric oxide synthase modulates the expression of molecular markers of muscle regeneration following hind limb ischemia reperfusion Bao-Ngoc H Nguyen, MD, Hassan Albadawi MD, Rabih Houbballah MD, Mounir J Haurani MD, Hyung-Jin Yoo MS Massachusetts General Hospital, Boston, MA INTRODUCTION: Inducible nitric oxide synthase (iNOS) expression during sepsis has been implicated in the development of muscle wasting. These experiments were designed to determine whether iNOS expression impedes skeletal muscle regeneration during the regenerative phase of ischemia reperfusion (IR) injury. METHODS: iNOS-null (iNOS⫺/⫺) and C57BL6 (C57) mice were subjected to 3hrs of hindlimb ischemia followed by reperfusion for 1, 7 or 14 days (D1, D7 and D14). Hindlimb function was analyzed using an injury scale; muscle protein poly-ADPribosylation (PAR, marker of PARP enzyme activity due to genomic and inflammatory stress), and myogenin (transcription factor associated with myofibers differentiation) expression were evaluated by immunoblotting. Total and phosphorylated-S473Akt were assessed using ELISA. Percent of injured or regenerating fibers were counted microscopically.
Surgical Forum Abstracts
S139
RESULTS: Both groups had equivalent skeletal muscle fiber injury (C57 87⫾5, iNOS⫺/⫺ 93⫾1, percent p⫽0.3) and functional deficit at D1 IR. Functional recovery was similar at D7 and D14. iNOS⫺/⫺ expressed transiently higher PAR at Day1, then had markedly lower levels at D7 and D14 IR. In contrast iNOS⫺/⫺ expressed higher myogenin levels throughout reperfusion (vs. C57). Muscle fiber morphology, total and phosphorylated-Akt were not different at any time point. Day 1
PAR (AU)
Day 14
iNOSⴚ/ⴚ
C57
iNOSⴚ/ⴚ
C57
4
4
3.5⫾0.2
3.6⫾0.15
2.2⫾0.1
2.1⫾0.2
25⫾ 4
76 ⫾15*
205⫾23
382⫾35*
138⫾35
349⫾12*
Limb Function Score Myogenin (AU)
Day 7
C57
iNOSⴚ/ⴚ
1519⫾183 2358⫾111* 1335⫾163 748⫾176* 843⫾152 484⫾152*
Total-Akt (pg/mg)
20⫾3
26⫾5
113⫾3
112⫾4
96⫾5
96⫾9
p473-Akt (units/mg)
4.1⫾0.3
5.2⫾0.3
28⫾2
31⫾2
27⫾5
23⫾3
*p⬍0.05 compared to C57
CONCLUSIONS: In these settings, iNOS deficiency decreased PARP enzyme activity and increased the level of myogenin in reperfused skeletal muscle. This suggest that iNOS is partially responsible for activation of PARP mediated inflammatory pathways, and suppresses myogenin expression during the regenerative phase of IR. However, iNOS dependent pathways do not appear to impede functional recovery or muscle fiber maturity.
Inhibition of prolyl hydroxylase attenuates microvascular inflammation after mesenteric ischemia/reperfusion Scott M Mullen MD, John G Wood PhD, Michael Moncure MD, James H Thomas MD University of Kansas Medical Center, Kansas City, KS INTRODUCTION: Acute mesenteric ischemia is a life-threatening condition that requires early intervention to improve blood flow. Restoration of blood flow to ischemic bowel results in ischemia/ reperfusion (I/R) injury, and the resulting microvascular inflammation contributes to the high morbidity and mortality associated with this condition. Inhibition of prolyl hydroxylase (PHD) increases hypoxiainducible factor-1 (HIF-1) levels, which promotes expression of genes affecting microvascular function. Our goal was to evaluate whether inhibition of PHD attenuated I/R-induced increases in leukocyte adherence/emigration and vascular permeability (VP), and if this involved upregulation of inducible nitric oxide synthase (iNOS). METHODS: Rats were injected with vehicle or the PHD inhibitor ethyl dihydrobenzoate (EDHB) 24 hr before experiments. Intravital microscopy was used to measure leukocyte adherence/emigration and VP in mesenteric venules of anesthetized rats. Mesentery was superfused with saline or the iNOS inhibitor L-NIL. After a control period, ischemia was produced by placing a microvascular clip on the superior mesenteric artery for 30 min, followed by reperfusion for 2 hrs. RESULTS: Data for leukocyte adherence are presented in the table. Similar results were obtained for leukocyte emigration and VP (data not shown). Leukocyte adherence/emigration and VP increased sig-
S140
Surgical Forum Abstracts
J Am Coll Surg
nificantly after I/R in vehicle-treated rats. However, EDHB significantly attenuated these responses after I/R. L-NIL significantly increased leukocyte adherence/emigration and VP during I/R in EDHB-treated rats. Adherent Leukocytes, # per 100 m venule
Time, min
Control, 15 30 Ischemia, 30 Reperfusion, 15 30 45 60 75 90 105 120
VehicleTreated (n ⴝ 5)
EDHBTreated (n ⴝ 5)
EDHBTreated ⴙ L-NIL (n ⴝ 5)
2.2 ⫾ 0.4 2.0 ⫾ 0.3 -7.0 ⫾ 0.9 7.4 ⫾ 0.5 8.8 ⫾ 0.9 10.2 ⫾ 0.6 10.4 ⫾ 1.1 10.4 ⫾ 1.9 11.0 ⫾ 1.2 12.4 ⫾ 2.9
1.7 ⫾ 0.3 1.7 ⫾ 0.4 -2.0 ⫾ 0.3 1.8 ⫾ 0.4* 2.2 ⫾ 0.4* 2.1 ⫾ 0.3* 2.4 ⫾ 0.4* 2.5 ⫾ 0.3* 2.1 ⫾ 0.3* 2.4 ⫾ 0.4*
1.6 ⫾ 0.3 1.8 ⫾ 0.3 -4.6 ⫾ 0.2 7.0 ⫾ 0.6 8.6 ⫾ 0.5† 8.2 ⫾ 0.4† 8.5 ⫾ 0.9† 8.6 ⫾ 0.6† 9.2 ⫾ 0.7† 9.8 ⫾ 0.8†
Values are means ⫾ standard errors. *indicates p ⬍ 0.05 vs corresponding vehicle-treated animals (ANOVA, specific contrasts). †indicates p ⬍ 0.05 vs corresponding EDHB-treated animals (ANOVA, specific contrasts).
CONCLUSIONS: Inhibition of PHD resulting in upregulation of HIF-1 attenuates microvascular inflammation following mesenteric I/R. This effect involves iNOS upregulation as inhibition of iNOS significantly increased leukocyte adherence/emigration and VP following I/R.
Toll-like receptor 4 mediates oxidized - LDL induced macrophage differentiation to foam cells Kenneth W Howell BS, Xianzhong Meng MD, PhD, David A Fullerton MD, FACS, Chunhua Jin PhD, Lihua Ao BS, T Brett Reece MD, Joseph C Cleveland Jr MD, FACS University of Colorado at Denver School of Medicine, Aurora, CO INTRODUCTION: Macrophage foam cells are central in the development of atherosclerosis but the mechanism of foam cell formation is unclear. Toll-like receptor 4 (TLR4) signaling is known to be important in the pathogenesis of atherosclerosis and oxidized LDL (oxLDL) enhances TLR4 expression in macrophages. We hypothesized that TLR4 is important in macrophage differentiation to foam cells. METHODS: Peritoneal macrophages were isolated by lavage from C3H/HeN (TLR4 competent) and C3H/HeJ (TLR4 dysfunctional) mice. Cells were treated with oxLDL, lipopolysaccharide (LPS) or oxidized LDL plus LPS. Cells were also treated with a TLR4 blocking antibody before oxLDL treatment. Oil red O staining for intracellular lipids was used for identification of foam cells. Statistical analysis was performed using ANOVA with Fisher’s post-test. RESULTS: TLR4 is necessary for macrophage differentiation to foam cells. There is a difference in the percentage of foam cell forma-
tion between TLR4 competent and TLR4 dysfunctional macrophages when treated with oxLDL. Following oxLDL treatment 29% of TLR4 competent cells differentiated to foam cells compared to 5.8% of TLR4 dysfunctional cells (p⬍0.01). Pre-treatment with a TLR4 blocking antibody decreased the differentiation of TLR4 competent cells to foam cells from 29% to 13% (p⬍0.01). Stimulation of TLR4 with LPS in the presence of oxLDL increased differentiation of TLR4 competent cells to foam cells from 29% to 60% (p⬍0.01). CONCLUSIONS: TLR4 is necessary for oxLDL-induced macrophage differentiation to foam cells. We conclude that TLR4 contributes to the pathogenesis of atherosclerosis by promoting foam cell formation.
Endothelial protective and anti-thrombotic effects of proanthocyanidins in a rat model of deep vein thrombosis Yunjian Zhang, Shenming Wang MD, PhD, FACS, Hanping Shi MD, PhD The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China INTRODUCTION: Inflammation plays a crucial role in endothelium injury and the progression of deep vein thrombosis (DVT). The present study was designed to investigate whether proanthocyanidins is a potential therapeutic agent that protects endothelium via the anti-inflammatory property. METHODS: Animal model was performed by the infrarenal inferior vena cava (IVC) ligation to induce DVT. After that, proanthocyanidins (400 mg/kg) was given to rats in the experimental group every 24 h. Vein wall endothelium and thrombi were observed by transmission electron microscopy (TEM). The IVC and iliac/femoral vein were processed for histological analysis. Related inflammatory markers were detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. RESULTS: Proanthocyanidins maintained the integrity of endothelium, reduced the size of thrombus, with up-regulated expression of CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells. Proanthocyanidins decreased the overexpression of von Willebrand factor (vWF) and increased a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) via the anti-inflammatory effects. In addition, the blood levels of inflammatory markers in experimental group were lower than the control (e.g., IL-6, IL-8, TNF-alpha, and LTB4). The expressions of monocyte chemotactic protein-1 (MCP-1), P-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1) were being modulated in experimental group. CONCLUSIONS: In conclusion, our study showed that treatment with proanthocyanidins provides benefit for the vein wall endothelium with the anti-inflammatory property to modulate the progress in thrombosis.