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Thrombosis-induced endothelial dysfunction is regulated by increased expression and activity of arginase
S114
Surgical Forum Abstracts
CONCLUSIONS: Inhaled CO inhibited platelet activation by thrombin and reduced platelet recruitment of neutrophils. These data demonstrate the ability of CO to block the initiating events involved in vascular injury and offers another potential mechanism by which inhaled CO may attenuate IH.
P53 activation accelerates resolution of deep vein thrombosis Lornie J Phillips II, MD, Erika Wong BS, Rajabrata Sarkar MD, PhD University of California, San Francisco; UCSF-East Bay, San Francisco, CA INTRODUCTION: The molecular mechanisms responsible for deep venous thrombosis resolution remain poorly understood. P53 is a transcription factor, previously noted to stimulate matrix metalloproteinase-2 expression, an enzyme important in thrombus resolution. As MMP2 knockout mice were recently found to have decreased thrombus resolution, we hypothesized that p53 would be an important signal in thrombus resolution. METHODS: DVT were generated by infrarenal caval ligation in mice lacking p53 and their littermate controls, as well as in CD1 mice treated with either continuous release quinacrine, a p53 activator, or its PBS vehicle starting 5 days prior to DVT generation. Thrombus weight was analyzed at day 3 and day 12, and expressed as thrombus weight/body weight ratios. RESULTS: Thrombi in p53 (⫺/⫺) mice were similar to their controls at day 3 (1.272 (mg/g) ⫾ 0.128, vs. 1.227 ⫾ 0.089. P⫽ 0.77. n⫽7) but were 31% larger than their controls on day 12 (0.386 ⫾ 0.0568 vs. 0.556 ⫾ 0.0412. P⫽0.021. n⫽10-13). Thrombi in CD1 mice that received quinacrine were similar to their PBS controls at day 3 (0.814 (mg/g) ⫾ 0.057, vs. 0.718 ⫾ 0.071. P⫽ 0.33. n⫽1013) but were 35% smaller than their controls on day 12 (0.355 ⫾ 0.0456 vs. 0.232 ⫾ 0.0319. P⫽0.04. n⫽6-9). CONCLUSIONS: P53 plays a critical role in thrombus resolution. These studies are the first to demonstrate both positive and negative regulation of thrombus resolution via modulation of the p53 pathway. Regulation of thrombus resolution by p53 increases our understanding of this clinically important process, and may provide favorable therapeutic targets.
Hyperspectral imaging: An emerging technology as a potential novel adjunct in assessing peripheral perfusion deficits and success of lower extremity revascularizations Owen N Johnson III MD, Mark Slidell MD, Peter Kreishman MD, Roger Walcott MD, James Scanlon MD, FACS, Subodh Arora MD, Robyn Macsata MD, FACS, Anton N Sidawy MD, FACS Washington DC Veterans Affairs Medical Center, Washington, DC INTRODUCTION: We investigated imaging spectroscopy as a possible adjunct in evaluating patients with lower extremity peripheral arterial disease (PAD) undergoing revascularization.
J Am Coll Surg
METHODS: This is a prospective study of a prototype hyperspectral imaging (HSI) device. Patients evaluated for PAD were included; images of dorsal and plantar surfaces of both feet were obtained across various wavelengths of light in addition to routine noninvasive tests and arteriography. Spectroscopic data were used to calculate capillary oxyhemoglobin (Hb-Oxy), deoxyhemoglobin (HbDeoxy), and oxygen saturation (O2-Sat) values. An infrared thermometer measured surface temperature. Revascularized patients were rescanned at predetermined intervals up to six weeks after surgery. Scan data were presented digitally in color-coded maps, allowing morphologic and quantitative comparisons. RESULTS: Eleven limbs in thirteen patients were revascularized, with 126 site measurements available for analysis. HSI successfully assessed the spatial distribution of cutaneous oxygenation, and all revascularized limbs demonstrated changes in their HSI profile. HbOxy surprisingly showed no statistically significant changes (P⫽.799). However, Hb-Deoxy decreased 40% (P⫽.009) and O2Sat levels increased 55% (P⫽.01). At each time point, control limbs showed no difference in Hb-Oxy (P⫽.425), Hb-Deoxy (P⫽.359), or O2-Sat (P⫽.568). Surface temperature was on average 8.1oF (⫹/⫺ 1.2oF) cooler in ischemic versus non-ischemic feet preoperatively, and equivalent postoperatively. In all thirteen patients, HSI profiles correlated with clinical course and arteriography findings. CONCLUSIONS: HSI is a novel, noninvasive method to qualitatively and quantitatively measure peripheral perfusion deficits and skin revascularization results over a heterogeneous spatial distribution. Patients with PAD do not have homogeneous foot skin envelope oxygenation even after successful revascularization.
Thrombosis-induced endothelial dysfunction is regulated by increased expression and activity of arginase Weifei Zhu PhD, Sydney M Morris PhD, Unni M Chandrasekharan PhD, Smarajit Bandyopadhyay PhD, Paul E DiCorleto PhD, Vikram S Kashyap MD, FACS The Cleveland Clinic, Cleveland, OH INTRODUCTION: Arterial thrombosis can lead to endothelial dysfunction and related vasospasm. Previous studies indicate thrombosis causes endothelial dysfunction without cell loss or affecting endothelial nitric oxide synthase (eNOS) enzyme activity. The objective of this study was to examine arginase, a competing enzyme for the eNOS substrate L-arginine, and possible mechanisms for endothelial dysfunction. METHODS: Rat aortic endothelial cells (RAECs) were exposed to thrombin at varying concentrations. Arginase protein activity was measured by the conversion of [14C] guanidinoarginine to [14C] urea. Transfection of RAECs with a 4.8 kb arginase I promoterLuciferase construct was performed to determine the mechanism by which thrombin upregulates arginase gene transcription. Different 5’ deletion mutants of the promoter complex were used to identify the region important for thrombin action.
Vol. 207, No. 3S, September 2008
RESULTS: Exposure of RAECs to thrombin upregulates arginase I mRNA and protein expression. Consistently, thrombin causes a 4.7 fold increase in arginase protein activity, with maximum activity reached at 4 hours (295⫹27 vs. 63⫹4 pmol urea/mg protein/hour, n⫽4, p⬍0.05). Thrombin strikingly increases arginase I promoter activity. Compared with control, 10 and 30 U/ml thrombin induced a 3.2 and 6.4 fold increase in luciferase activity respectively (n⫽3, p⬍0.05 versus control). Using different 5’ deletion mutants, the region between ⫺3.29 kb and ⫺2.78 kb in the arginase I promoter complex is identified as important for thrombin action. CONCLUSIONS: Arterial thrombosis-induced endothelial dysfunction is caused by increased arginase protein activity via upregulation of gene expression. Arginase blockade may prove to be an attractive therapeutic alternative for endothelial dysfunction.
Injection of the anti-angiogenic, hemopexin-like fragment (PEX2) of matrix metalloproteinase-2 (MMP-2) induces delayed wound healing April E Nedeau MD, Katherine Gallagher MD, Omaida C Velazquez MD, FACS University of Pennsylvania, Philadelphia, PA INTRODUCTION: Matrix metalloproteinase-2(MMP-2) degrades type IV collagen permitting endothelial cell(EC) migration through extracellular matrix during wound angiogenesis. PEX2, a byproduct of MMP-2 degradation, competitively inhibits newly activated MMP-2 from EC surface binding and migration, effectively reducing angiogenesis. We have shown that PEX2 levels increase in ischemic murine hindlimbs and hypothesize that PEX2 elevation in wounds results in delayed healing. METHODS: FVB mice were wounded with a 6mm punch biopsy on their dorsum. 75pmol recombinant PEX2 protein was injected into the wounds daily versus injection of saline. Wounds were photographed daily. The area of exposed muscle, as seen through the gap in granulation tissue, was measured using NIH Image J software and calculated as percent healing per day. Wounds were harvested on postoperative day 7, fixed with Prefer, embedded in paraffin and serially sectioned for staining with hematoxylin and eosin. Epithelial gap was measured via microscopy and Image J software. RESULTS: Healing was reduced in the PEX2 group by 6 to 16% over the first postoperative week (p⬍0.02). These wounds were characterized by poor granulation tissue formation and exposed muscle in the center, while the control wounds were completely covered with granulation tissue by POD 5. When epithelial gap was measured, the diameter of the PEX2 wounds was more then two fold greater then the control (p⬍0.05). CONCLUSIONS: Elevation of wound level PEX2 significantly reduces the healing rate, as measured by exposed muscle, granulation tissue formation and epithelial gap. Neutralizing PEX2 within wounds may be a novel, potentially therapeutic target in the treatment of ischemic wounds.
Surgical Forum Abstracts
S115
Skeletal muscle functional recovery in the hyperlipidemic murine model of human vascular disease Faraz F Hashmi AB, Hassan Albadawi MD, James B Kobler PhD, James T Heaton PhD, Jeanwan Kang MD, Hyung-Jin Yoo MA, Michael Peck MD, Michael T Watkins MD, FACS Massachusetts General Hospital, Boston, MA INTRODUCTION: While the biochemical and inflammatory components of experimental acute hind limb ischemia reperfusion are well described, little is known about quantitative aspects of skeletal muscle functional recovery in murine models of human vascular disease. Experiments were designed to determine muscle contractile force during the chronic phases of reperfusion injury in the hyperlipidemic murine model. METHODS: ApolipoproteinE deficient retired-breeders (ApoE⫺/⫺, n⫽5-7) and age-matched wild-type controls (C57BL/6, n⫽5-7) were subjected to hind-limb ischemia for 1.5 hours ischemia followed by 4 and 6-weeks reperfusion (IR). Limbs were subjected to direct electrical muscle stimulation in vivo to assess peak contractileforce as an index of functional recovery during reperfusion. Nonischemic sham animals served as baseline. ANOVA was used for comparisons. RESULTS: In C57BL/6, there was significantly less contractileforce by 4-weeks IR versus sham (34⫾2 vs. 52⫾1.1 grams, p⬍0.05). By 6-weeks, IR contractile force recovered close to baseline and was not statistically significantly different from sham (45⫾3 vs. 52⫾1.1 grams). In a similar manner, ApoE⫺/⫺ displayed significantly less force than sham by 4 weeks reperfusion (30⫾3 vs. 61⫾4 grams, p⬍0.001). However, by 6-weeks IR, ApoE⫺/⫺ mice continue to have less force than their sham (36⫾1 vs. 61⫾4 grams p⫽0.001). CONCLUSIONS: Contractile force in C57BL/6 mice gradually recovered to sham levels by 6-weeks IR. In contrast, ApoE⫺/⫺ displays significantly less contractile force at 6-weeks IR versus sham. This suggests that the hyperlipidemic phenotype impairs functional recovery following hind limb ischemia reperfusion, which may be an important component of limb humans recovering from acute limb ischemia reperfusion.
Heme oxygenase upregulation contributes to microvascular acclimatization to chronic hypoxia Alfred J Casillan MD, PhD, John G Wood PhD, Norberto C Gonzalez MD, Michael Moncure MD, FACS, James H Thomas MD, FACS University of Kansas Medical Center, Kansas City, KS INTRODUCTION: Systemic hypoxia (Hx) initiates rapid microvascular inflammation in rats that is characterized by increased leukocyte adherence, leukocyte emigration, and venular permeability. During 3 weeks of Hx, this inflammation resolves and the microcirculation becomes resistant to further decreases in inspired O2. This study investigated the potential role of heme oxygenase (HO) in microvascular acclimatization to chronic Hx.
Surgical Forum Abstracts
CONCLUSIONS: Inhaled CO inhibited platelet activation by thrombin and reduced platelet recruitment of neutrophils. These data demonstrate the ability of CO to block the initiating events involved in vascular injury and offers another potential mechanism by which inhaled CO may attenuate IH.
P53 activation accelerates resolution of deep vein thrombosis Lornie J Phillips II, MD, Erika Wong BS, Rajabrata Sarkar MD, PhD University of California, San Francisco; UCSF-East Bay, San Francisco, CA INTRODUCTION: The molecular mechanisms responsible for deep venous thrombosis resolution remain poorly understood. P53 is a transcription factor, previously noted to stimulate matrix metalloproteinase-2 expression, an enzyme important in thrombus resolution. As MMP2 knockout mice were recently found to have decreased thrombus resolution, we hypothesized that p53 would be an important signal in thrombus resolution. METHODS: DVT were generated by infrarenal caval ligation in mice lacking p53 and their littermate controls, as well as in CD1 mice treated with either continuous release quinacrine, a p53 activator, or its PBS vehicle starting 5 days prior to DVT generation. Thrombus weight was analyzed at day 3 and day 12, and expressed as thrombus weight/body weight ratios. RESULTS: Thrombi in p53 (⫺/⫺) mice were similar to their controls at day 3 (1.272 (mg/g) ⫾ 0.128, vs. 1.227 ⫾ 0.089. P⫽ 0.77. n⫽7) but were 31% larger than their controls on day 12 (0.386 ⫾ 0.0568 vs. 0.556 ⫾ 0.0412. P⫽0.021. n⫽10-13). Thrombi in CD1 mice that received quinacrine were similar to their PBS controls at day 3 (0.814 (mg/g) ⫾ 0.057, vs. 0.718 ⫾ 0.071. P⫽ 0.33. n⫽1013) but were 35% smaller than their controls on day 12 (0.355 ⫾ 0.0456 vs. 0.232 ⫾ 0.0319. P⫽0.04. n⫽6-9). CONCLUSIONS: P53 plays a critical role in thrombus resolution. These studies are the first to demonstrate both positive and negative regulation of thrombus resolution via modulation of the p53 pathway. Regulation of thrombus resolution by p53 increases our understanding of this clinically important process, and may provide favorable therapeutic targets.
Hyperspectral imaging: An emerging technology as a potential novel adjunct in assessing peripheral perfusion deficits and success of lower extremity revascularizations Owen N Johnson III MD, Mark Slidell MD, Peter Kreishman MD, Roger Walcott MD, James Scanlon MD, FACS, Subodh Arora MD, Robyn Macsata MD, FACS, Anton N Sidawy MD, FACS Washington DC Veterans Affairs Medical Center, Washington, DC INTRODUCTION: We investigated imaging spectroscopy as a possible adjunct in evaluating patients with lower extremity peripheral arterial disease (PAD) undergoing revascularization.
J Am Coll Surg
METHODS: This is a prospective study of a prototype hyperspectral imaging (HSI) device. Patients evaluated for PAD were included; images of dorsal and plantar surfaces of both feet were obtained across various wavelengths of light in addition to routine noninvasive tests and arteriography. Spectroscopic data were used to calculate capillary oxyhemoglobin (Hb-Oxy), deoxyhemoglobin (HbDeoxy), and oxygen saturation (O2-Sat) values. An infrared thermometer measured surface temperature. Revascularized patients were rescanned at predetermined intervals up to six weeks after surgery. Scan data were presented digitally in color-coded maps, allowing morphologic and quantitative comparisons. RESULTS: Eleven limbs in thirteen patients were revascularized, with 126 site measurements available for analysis. HSI successfully assessed the spatial distribution of cutaneous oxygenation, and all revascularized limbs demonstrated changes in their HSI profile. HbOxy surprisingly showed no statistically significant changes (P⫽.799). However, Hb-Deoxy decreased 40% (P⫽.009) and O2Sat levels increased 55% (P⫽.01). At each time point, control limbs showed no difference in Hb-Oxy (P⫽.425), Hb-Deoxy (P⫽.359), or O2-Sat (P⫽.568). Surface temperature was on average 8.1oF (⫹/⫺ 1.2oF) cooler in ischemic versus non-ischemic feet preoperatively, and equivalent postoperatively. In all thirteen patients, HSI profiles correlated with clinical course and arteriography findings. CONCLUSIONS: HSI is a novel, noninvasive method to qualitatively and quantitatively measure peripheral perfusion deficits and skin revascularization results over a heterogeneous spatial distribution. Patients with PAD do not have homogeneous foot skin envelope oxygenation even after successful revascularization.
Thrombosis-induced endothelial dysfunction is regulated by increased expression and activity of arginase Weifei Zhu PhD, Sydney M Morris PhD, Unni M Chandrasekharan PhD, Smarajit Bandyopadhyay PhD, Paul E DiCorleto PhD, Vikram S Kashyap MD, FACS The Cleveland Clinic, Cleveland, OH INTRODUCTION: Arterial thrombosis can lead to endothelial dysfunction and related vasospasm. Previous studies indicate thrombosis causes endothelial dysfunction without cell loss or affecting endothelial nitric oxide synthase (eNOS) enzyme activity. The objective of this study was to examine arginase, a competing enzyme for the eNOS substrate L-arginine, and possible mechanisms for endothelial dysfunction. METHODS: Rat aortic endothelial cells (RAECs) were exposed to thrombin at varying concentrations. Arginase protein activity was measured by the conversion of [14C] guanidinoarginine to [14C] urea. Transfection of RAECs with a 4.8 kb arginase I promoterLuciferase construct was performed to determine the mechanism by which thrombin upregulates arginase gene transcription. Different 5’ deletion mutants of the promoter complex were used to identify the region important for thrombin action.
Vol. 207, No. 3S, September 2008
RESULTS: Exposure of RAECs to thrombin upregulates arginase I mRNA and protein expression. Consistently, thrombin causes a 4.7 fold increase in arginase protein activity, with maximum activity reached at 4 hours (295⫹27 vs. 63⫹4 pmol urea/mg protein/hour, n⫽4, p⬍0.05). Thrombin strikingly increases arginase I promoter activity. Compared with control, 10 and 30 U/ml thrombin induced a 3.2 and 6.4 fold increase in luciferase activity respectively (n⫽3, p⬍0.05 versus control). Using different 5’ deletion mutants, the region between ⫺3.29 kb and ⫺2.78 kb in the arginase I promoter complex is identified as important for thrombin action. CONCLUSIONS: Arterial thrombosis-induced endothelial dysfunction is caused by increased arginase protein activity via upregulation of gene expression. Arginase blockade may prove to be an attractive therapeutic alternative for endothelial dysfunction.
Injection of the anti-angiogenic, hemopexin-like fragment (PEX2) of matrix metalloproteinase-2 (MMP-2) induces delayed wound healing April E Nedeau MD, Katherine Gallagher MD, Omaida C Velazquez MD, FACS University of Pennsylvania, Philadelphia, PA INTRODUCTION: Matrix metalloproteinase-2(MMP-2) degrades type IV collagen permitting endothelial cell(EC) migration through extracellular matrix during wound angiogenesis. PEX2, a byproduct of MMP-2 degradation, competitively inhibits newly activated MMP-2 from EC surface binding and migration, effectively reducing angiogenesis. We have shown that PEX2 levels increase in ischemic murine hindlimbs and hypothesize that PEX2 elevation in wounds results in delayed healing. METHODS: FVB mice were wounded with a 6mm punch biopsy on their dorsum. 75pmol recombinant PEX2 protein was injected into the wounds daily versus injection of saline. Wounds were photographed daily. The area of exposed muscle, as seen through the gap in granulation tissue, was measured using NIH Image J software and calculated as percent healing per day. Wounds were harvested on postoperative day 7, fixed with Prefer, embedded in paraffin and serially sectioned for staining with hematoxylin and eosin. Epithelial gap was measured via microscopy and Image J software. RESULTS: Healing was reduced in the PEX2 group by 6 to 16% over the first postoperative week (p⬍0.02). These wounds were characterized by poor granulation tissue formation and exposed muscle in the center, while the control wounds were completely covered with granulation tissue by POD 5. When epithelial gap was measured, the diameter of the PEX2 wounds was more then two fold greater then the control (p⬍0.05). CONCLUSIONS: Elevation of wound level PEX2 significantly reduces the healing rate, as measured by exposed muscle, granulation tissue formation and epithelial gap. Neutralizing PEX2 within wounds may be a novel, potentially therapeutic target in the treatment of ischemic wounds.
Surgical Forum Abstracts
S115
Skeletal muscle functional recovery in the hyperlipidemic murine model of human vascular disease Faraz F Hashmi AB, Hassan Albadawi MD, James B Kobler PhD, James T Heaton PhD, Jeanwan Kang MD, Hyung-Jin Yoo MA, Michael Peck MD, Michael T Watkins MD, FACS Massachusetts General Hospital, Boston, MA INTRODUCTION: While the biochemical and inflammatory components of experimental acute hind limb ischemia reperfusion are well described, little is known about quantitative aspects of skeletal muscle functional recovery in murine models of human vascular disease. Experiments were designed to determine muscle contractile force during the chronic phases of reperfusion injury in the hyperlipidemic murine model. METHODS: ApolipoproteinE deficient retired-breeders (ApoE⫺/⫺, n⫽5-7) and age-matched wild-type controls (C57BL/6, n⫽5-7) were subjected to hind-limb ischemia for 1.5 hours ischemia followed by 4 and 6-weeks reperfusion (IR). Limbs were subjected to direct electrical muscle stimulation in vivo to assess peak contractileforce as an index of functional recovery during reperfusion. Nonischemic sham animals served as baseline. ANOVA was used for comparisons. RESULTS: In C57BL/6, there was significantly less contractileforce by 4-weeks IR versus sham (34⫾2 vs. 52⫾1.1 grams, p⬍0.05). By 6-weeks, IR contractile force recovered close to baseline and was not statistically significantly different from sham (45⫾3 vs. 52⫾1.1 grams). In a similar manner, ApoE⫺/⫺ displayed significantly less force than sham by 4 weeks reperfusion (30⫾3 vs. 61⫾4 grams, p⬍0.001). However, by 6-weeks IR, ApoE⫺/⫺ mice continue to have less force than their sham (36⫾1 vs. 61⫾4 grams p⫽0.001). CONCLUSIONS: Contractile force in C57BL/6 mice gradually recovered to sham levels by 6-weeks IR. In contrast, ApoE⫺/⫺ displays significantly less contractile force at 6-weeks IR versus sham. This suggests that the hyperlipidemic phenotype impairs functional recovery following hind limb ischemia reperfusion, which may be an important component of limb humans recovering from acute limb ischemia reperfusion.
Heme oxygenase upregulation contributes to microvascular acclimatization to chronic hypoxia Alfred J Casillan MD, PhD, John G Wood PhD, Norberto C Gonzalez MD, Michael Moncure MD, FACS, James H Thomas MD, FACS University of Kansas Medical Center, Kansas City, KS INTRODUCTION: Systemic hypoxia (Hx) initiates rapid microvascular inflammation in rats that is characterized by increased leukocyte adherence, leukocyte emigration, and venular permeability. During 3 weeks of Hx, this inflammation resolves and the microcirculation becomes resistant to further decreases in inspired O2. This study investigated the potential role of heme oxygenase (HO) in microvascular acclimatization to chronic Hx.