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reperfusion induced microvascular injury in vivo
Surgical Forum Abstracts
Vascular Surgery I
subsequent transmigration of leucocytes is the pivotal step in the inflammatory response. We hypothesised that pravastatin, a HMGcoA inhibitor, would attenuate the inflammatory response and tested this hypothesis in a model of I/R-induced inflammatory response in a normo-lipidaemic rat using intravital microscopy.
Simvastatin induces apoptosis in proliferating human saphenous vein endothelial cells through PARP cleavage
Methods: Sprague-Dawley rats were randomised into control (sham I/R), I/R and pravastatin ⫹ I/R groups (Pra ⫹ IR). Pravastatin (4 mg/kg body weight/day) or saline was administrated by oral gavage for 4 days before I/R injury. I/R injury was established by occlusion of the superior mesenteric and coeliac vascular pedicle for 20 min, followed by 60 min of reperfusion. Leucocyte rolling velocity, the number of adherent and migrated leucocytes were measured in postcapillary venules at baseline (⫺10 min) and 10, 30, 60 min after reperfusion. 5 rats were excluded because of no-reflow after ischaemia (3/8 rats in I/R and 2/8 rats in pravastatin ⫹ I/R group). I/R significantly decreased the rolling velocity of leucocytes at 10, 30, 60 min after reperfusion, and increased the number of adherent and migrated leucocyte at 30 and 60 min after reperfusion. Pravastatin significantly attenuated these changes after reperfusion.
R. Brannon Claytor, MD, Jian-Ming Li, MD, Shimul Shah, MD, John Herrmann, MD, Robert C Harland, MD, Michael J Rohrer, MD. Department of Surgery, Division of Vascular Surgery, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, USA (508) 856-3537. Introduction: Simvastatin, a potent HMG-CoA reductase inhibitor used clinically to lower serum cholesterol, has been shown to inhibit proliferation of endothelial cells. The mechanism through which this effect is mediated is not well characterized. We hypothesize that simvastatin induces apoptosis of proliferating human saphenous vein endothelial cells (HSVECs). Methods: HSVECs were seeded into 6-well plates and cultured for 48 hrs. The proliferating cells were then incubated for an additional 48 hrs with either buffer alone, 20 M simvastatin, or 50 ng/mL TNF-␣. A viable cell count was determined by trypan blue exclusion. Apoptosis was evaluated by nuclear morphology (H&E stain), DNA fragmentation (APO-BRDU assay), annexin V binding, and cleavage of the caspase substrate PARP by Western blot. Statistical analysis- two-tailed t-test.
Results: Results at 60 min reperfusion
Control (n ⴝ 6)
Rolling velocity (m/sec) 48.37 ⫾ 1.82 No. adherence (/100m) 2.00 ⫾ 0.52 No. migrated (/field) 2.50 ⫾ 0.56
Results: Proliferation of HSVECs following a 48 hr incubation with simvastatin (2.85 ⫻ 106 ⫾ 1.4 ⫻ 106, mean ⫾ SEM, n ⫽ 4) or TNF-␣ (2.1 ⫻ 106 ⫾ 0.9 ⫻ 106, n ⫽ 4) was significantly reduced compared to buffer alone (10.4 ⫻ 106 ⫾ 0.9 ⫻ 106, n ⫽ 4) (p ⬍ 0.05). HSVECs treated with TNF-␣ or simvastatin demonstrated morphologic changes of apoptosis and had a 5-fold increase in annexin V binding compared to buffer treated cells (data not shown). Additionally, the APO-BRDU DNA fragmentation assay (Figure 1) demonstrated a significant increase in apoptosis when the cells were treated with simvastatin or TNF-␣. Western blot analysis demonstrated increased cleavage of PARP, a common final pathway component of apoptosis, for both simvastatin and TNF-␣ (Figure 2).
I/R (n ⴝ 5)
Pra ⴙ I/R (n ⴝ 6)
33.30 ⫾ 3.41* £ 22.20 ⫾ 3.43* £ 9.60 ⫾ 1.33* ££
46.04 ⫾ 1.76 3.17 ⫾ 0.79 4.00 ⫾ 1.34
Mean ⫾ SEM, Stat: ANOVA with Scheffe´ post hoc test, *: P ⬎ 0.01 vs control, and £: P ⬎ 0.01 and ££: P ⬎ 0.05 vs pravastatin ⫹ I/R.
Conclusions: These data demonstrated that pravastatin attenuated I/R-induced microvascular injury by inhibiting leucocyte adhesion and migration, and this protection may be independent of its cholesterollowering effect. The mechanism by which pravastatin decreased leucocyte adhesion and migration is unknown.
VEGF activates nuclear factor of activated T-cells (NFAT) but not NF-B in human microvascular endothelial cells Parvaneh Rafiee, PhD1, David Binion, MD2, Mona Li, MD1, Cara Olds, MS1, Mark Adams, MD1, Christopher Johnson, MD1. Med. Coll. of Wisconsin, Dept. of Surgery1 and Medicine2, 9200 W. Wisconsin Avenue, Milwaukee, WI 53226, USA. (414) 456-4666.
Figure 1: Apoptosis of HSVECs following 48 hr incubation, (n ⴝ 4), *p < 0.05
Introduction: Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis, vascular permeability and monocyte recruitment in health and during pathological conditions, including chronic inflammation and tumor neo-vascularization. The molecular mechanisms and signal transduction cascades leading to the multiple biological functions of VEGF are largely unknown. The aim of this study was to investigate the effect of VEGF on signaling pathways in human intestinal microvascular endothelial cells (EC) and to determine the role of NFAT and nuclear factor-kappa B (NF-B) in VEGF mediated cell growth and leukocyte adhesion.
Figure 2: Western blot of PARP antibody at 86 kd Spot density
Conclusions: Simvastatin inhibits HSVECs’ proliferation by inducing apoptosis. The mechanism for apoptosis involves PARP-cleavage, which is shared by TNF-␣.
Gang Chen MD, Dana Moneley FRCSI, Cathal Kelly FRCSI, Hong Chen MD, Austin Leahy FRCSI, David Bouchier-Hayes FRCSI. Royal College of Surgeons in Ireland, Department of Surgery, Beaumont Hospital, Dublin 9, Ireland, 353-1-8377755, ext. 2680.
Methods: Primary cultures of EC were used in all experiments. Intracellular signaling pathways were studied in unstimulated and VEGFactivated EC monolayers. EC were pretreated with MAPK inhibitors (PD 98059, SB 203580, curcumin), NF-B inhibitor (Bay11), or calcineurin inhibitor cyclosporin A (CsA) prior to VEGF stimulation. Nuclear and cytosolic extracts were analyzed by SDS-PAGE/western blotting and EMSA. EC growth and proliferation were quantified using Coulter Counter. Leukocyte binding was quantified using a U-937-EC adhesion assay.
Introduction: The ability of HMGcoA inhibitor to improve prognosis in cardio-vascular disease is not solely due to the lowering of lipid. It has been suggested that these agents are also anti-inflammatory. However, this effect has not been confirmed in vivo. The adhesion and
Results: EC proliferated in response to VEGF (double vs. control). Unstimulated EC bound low levels of U-937 monocytes, which increased 10 fold following VEGF activation. VEGF stimulation resulted in the activation of p42/44 MAPK and nuclear translocation of NFAT
Pravastatin protects against ischaemia/reperfusion induced microvascular injury in vivo
© 2000 by the American College of Surgeons Published by Elsevier Science Inc.
S1
ISSN 1072-7515/00/$21.00
Vascular Surgery I
subsequent transmigration of leucocytes is the pivotal step in the inflammatory response. We hypothesised that pravastatin, a HMGcoA inhibitor, would attenuate the inflammatory response and tested this hypothesis in a model of I/R-induced inflammatory response in a normo-lipidaemic rat using intravital microscopy.
Simvastatin induces apoptosis in proliferating human saphenous vein endothelial cells through PARP cleavage
Methods: Sprague-Dawley rats were randomised into control (sham I/R), I/R and pravastatin ⫹ I/R groups (Pra ⫹ IR). Pravastatin (4 mg/kg body weight/day) or saline was administrated by oral gavage for 4 days before I/R injury. I/R injury was established by occlusion of the superior mesenteric and coeliac vascular pedicle for 20 min, followed by 60 min of reperfusion. Leucocyte rolling velocity, the number of adherent and migrated leucocytes were measured in postcapillary venules at baseline (⫺10 min) and 10, 30, 60 min after reperfusion. 5 rats were excluded because of no-reflow after ischaemia (3/8 rats in I/R and 2/8 rats in pravastatin ⫹ I/R group). I/R significantly decreased the rolling velocity of leucocytes at 10, 30, 60 min after reperfusion, and increased the number of adherent and migrated leucocyte at 30 and 60 min after reperfusion. Pravastatin significantly attenuated these changes after reperfusion.
R. Brannon Claytor, MD, Jian-Ming Li, MD, Shimul Shah, MD, John Herrmann, MD, Robert C Harland, MD, Michael J Rohrer, MD. Department of Surgery, Division of Vascular Surgery, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, USA (508) 856-3537. Introduction: Simvastatin, a potent HMG-CoA reductase inhibitor used clinically to lower serum cholesterol, has been shown to inhibit proliferation of endothelial cells. The mechanism through which this effect is mediated is not well characterized. We hypothesize that simvastatin induces apoptosis of proliferating human saphenous vein endothelial cells (HSVECs). Methods: HSVECs were seeded into 6-well plates and cultured for 48 hrs. The proliferating cells were then incubated for an additional 48 hrs with either buffer alone, 20 M simvastatin, or 50 ng/mL TNF-␣. A viable cell count was determined by trypan blue exclusion. Apoptosis was evaluated by nuclear morphology (H&E stain), DNA fragmentation (APO-BRDU assay), annexin V binding, and cleavage of the caspase substrate PARP by Western blot. Statistical analysis- two-tailed t-test.
Results: Results at 60 min reperfusion
Control (n ⴝ 6)
Rolling velocity (m/sec) 48.37 ⫾ 1.82 No. adherence (/100m) 2.00 ⫾ 0.52 No. migrated (/field) 2.50 ⫾ 0.56
Results: Proliferation of HSVECs following a 48 hr incubation with simvastatin (2.85 ⫻ 106 ⫾ 1.4 ⫻ 106, mean ⫾ SEM, n ⫽ 4) or TNF-␣ (2.1 ⫻ 106 ⫾ 0.9 ⫻ 106, n ⫽ 4) was significantly reduced compared to buffer alone (10.4 ⫻ 106 ⫾ 0.9 ⫻ 106, n ⫽ 4) (p ⬍ 0.05). HSVECs treated with TNF-␣ or simvastatin demonstrated morphologic changes of apoptosis and had a 5-fold increase in annexin V binding compared to buffer treated cells (data not shown). Additionally, the APO-BRDU DNA fragmentation assay (Figure 1) demonstrated a significant increase in apoptosis when the cells were treated with simvastatin or TNF-␣. Western blot analysis demonstrated increased cleavage of PARP, a common final pathway component of apoptosis, for both simvastatin and TNF-␣ (Figure 2).
I/R (n ⴝ 5)
Pra ⴙ I/R (n ⴝ 6)
33.30 ⫾ 3.41* £ 22.20 ⫾ 3.43* £ 9.60 ⫾ 1.33* ££
46.04 ⫾ 1.76 3.17 ⫾ 0.79 4.00 ⫾ 1.34
Mean ⫾ SEM, Stat: ANOVA with Scheffe´ post hoc test, *: P ⬎ 0.01 vs control, and £: P ⬎ 0.01 and ££: P ⬎ 0.05 vs pravastatin ⫹ I/R.
Conclusions: These data demonstrated that pravastatin attenuated I/R-induced microvascular injury by inhibiting leucocyte adhesion and migration, and this protection may be independent of its cholesterollowering effect. The mechanism by which pravastatin decreased leucocyte adhesion and migration is unknown.
VEGF activates nuclear factor of activated T-cells (NFAT) but not NF-B in human microvascular endothelial cells Parvaneh Rafiee, PhD1, David Binion, MD2, Mona Li, MD1, Cara Olds, MS1, Mark Adams, MD1, Christopher Johnson, MD1. Med. Coll. of Wisconsin, Dept. of Surgery1 and Medicine2, 9200 W. Wisconsin Avenue, Milwaukee, WI 53226, USA. (414) 456-4666.
Figure 1: Apoptosis of HSVECs following 48 hr incubation, (n ⴝ 4), *p < 0.05
Introduction: Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis, vascular permeability and monocyte recruitment in health and during pathological conditions, including chronic inflammation and tumor neo-vascularization. The molecular mechanisms and signal transduction cascades leading to the multiple biological functions of VEGF are largely unknown. The aim of this study was to investigate the effect of VEGF on signaling pathways in human intestinal microvascular endothelial cells (EC) and to determine the role of NFAT and nuclear factor-kappa B (NF-B) in VEGF mediated cell growth and leukocyte adhesion.
Figure 2: Western blot of PARP antibody at 86 kd Spot density
Conclusions: Simvastatin inhibits HSVECs’ proliferation by inducing apoptosis. The mechanism for apoptosis involves PARP-cleavage, which is shared by TNF-␣.
Gang Chen MD, Dana Moneley FRCSI, Cathal Kelly FRCSI, Hong Chen MD, Austin Leahy FRCSI, David Bouchier-Hayes FRCSI. Royal College of Surgeons in Ireland, Department of Surgery, Beaumont Hospital, Dublin 9, Ireland, 353-1-8377755, ext. 2680.
Methods: Primary cultures of EC were used in all experiments. Intracellular signaling pathways were studied in unstimulated and VEGFactivated EC monolayers. EC were pretreated with MAPK inhibitors (PD 98059, SB 203580, curcumin), NF-B inhibitor (Bay11), or calcineurin inhibitor cyclosporin A (CsA) prior to VEGF stimulation. Nuclear and cytosolic extracts were analyzed by SDS-PAGE/western blotting and EMSA. EC growth and proliferation were quantified using Coulter Counter. Leukocyte binding was quantified using a U-937-EC adhesion assay.
Introduction: The ability of HMGcoA inhibitor to improve prognosis in cardio-vascular disease is not solely due to the lowering of lipid. It has been suggested that these agents are also anti-inflammatory. However, this effect has not been confirmed in vivo. The adhesion and
Results: EC proliferated in response to VEGF (double vs. control). Unstimulated EC bound low levels of U-937 monocytes, which increased 10 fold following VEGF activation. VEGF stimulation resulted in the activation of p42/44 MAPK and nuclear translocation of NFAT
Pravastatin protects against ischaemia/reperfusion induced microvascular injury in vivo
© 2000 by the American College of Surgeons Published by Elsevier Science Inc.
S1
ISSN 1072-7515/00/$21.00