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Effect of decellularization protocol of human saphenous veins on cytotoxicity and matrix component
Abstracts / Atherosclerosis 244 (2016) e1ee12
The proven involvement of adaptive immunity, coupled with a phenotypic switch from autoimmune suppressive tolerogenic Treg to pro-inflammatory CD4+ Th1 and CD8+ T cells, during progression of obesity necessitates the presence of a triggering antigen as an activator of T and B cells. HSP60 is an evolutionary conserved mitochondrial chaperonin which can be located in the cytosol or exposed on the cell membrane also. An increase in cell membrane HSP60, which may be accompanied by HSP60 release into circulation, is especially considered a signal of autoimmunity. HSP60 is linked to obesity as a mediator of adipose tissue inflammation and insulin resistance. Moreover, circulating HSP60 levels are found to be higher in obese individuals than lean controls1. We observed an adaptive immune response against HSP60 at both T cell and B cell (antibody) levels during continuous high fat feeding of C57bl6 mice. Hence, HSP60 appears to be one of the mystery auto-antigens triggering the early T and B cell responses during obesity. Furthermore, we attempted a peptide therapy in a dose escalation protocol aiming to downregulate the inflammatory related adverse effects of obesity by achieving tolerance in T cell populations and suppressing the pathogenic antibody response. Using previously defined immuno-dominant murine HSP60 peptide combinations, we observed a significant improvement in insulin resistance (p ¼ 0.002, 2-way ANOVA) and circulating LDL/VLDL level (p ¼ 0.028). We also observed some improvement in glucose tolerance at borderline significance (p ¼ 0.057), which encourages further research to improve peptide therapy. 1 € €de A, Kriebel J, Ouwens DM, et al. Heat Marker T, Sell H, Zillessen P, Glo shock protein 60 as a mediator of adipose tissue inflammation and insulin resistance. Diabetes. 2012;61(3):615e25.
THE PLANT EXTRACT MOMORDICA CHARANTIA REDUCES EXPRESSION OF VASCULAR CALCIFICATION MARKERS IN A SMOOTH MUSCLE CELL CULTURE MODEL G.P. Sidgwick 1, *, R. Weston 1, N. Ahmad 1, N. Nazhad 1, A. Schiro 2, 3, F. Serracino Inglott 1, 2, 3, F.L. Wilkinson 1, M.Y. Alexander 1, 2. 1 Healthcare Science Research Centre, Manchester Metropolitan University, Chester Street, Manchester, M1 5GD, UK; 2 Cardiovascular Research Institute, University of Manchester, Grafton St, Manchester, Manchester Academic Health Science Centre, UK; 3 Vascular Unit, Central Manchester NHS Trust, Manchester Royal Infirmary, UK * Presenting author. The onset of vascular calcification in type-2 diabetic patients is one of the major health burdens in the industrialised world due to the associated increase in comorbidities and mortality. The fruits of the subtropical vine Momordica Charantia are thought to have potent anti-diabetic effects due to the anti-hyperglycaemic, anti-oxidant and anti-inflammatory properties of its 200+ constituents, some of which may mimic the effect of insulin. The aim of this study was to investigate the inhibitory effect of Momordica Charantia extract on vascular calcification pathways in a cell culture model. Vascular smooth muscle cells (VSMC) were isolated and cultured from femoral arteries following amputation from diabetic patients. Osteogenic differentiation was induced using b-glycerophosphate (b-GP), methylglyoxal (MGO) and additional advanced glycated end-products (AGEs). The cells were subsequently treated with varying concentrations of Momordica Charantia extract to determine the anti-calcification effects in vitro using qPCR, Alkaline phosphatase (ALP) activity and Alizarin red staining. We demonstrate that Momordica Charantia reduced gene expression of a range of biomarkers linked with vascular calcification after 4 days in a dose-dependant manner, including osteocalcin, BMP-2, C-MET and NOX-1, compared with osteogenic controls. ALP activity was also reduced in treated cells compared with osteogenic controls. Momordica Charantia extract shows promise as a potential therapeutic intervention to reduce calcification. Several small-scale clinical studies have shown the benefit of dietary supplementation in reducing the effects of diabetes, however further work is required to identify the specific component responsible for the effects, elucidate the mechanisms involved and validate the response in vitro.
e11
CYTOSKELETAL REMODELLING AND INHIBITION OF MKL DEPENDENT TRANSCRIPTION UNDERLIES THE ANTI-MITOGENIC EFFECTS OF CAMP IN SMOOTH MUSCLE CELLS M.C. Smith*, A. Duggirala, T.E. Kimura, G.B. Newby, A.C. Newby, M. Bond. School of Clinical Sciences, University of Bristol, UK * Presenting author. Background: Vascular smooth muscle cell (VSMC) proliferation contributes to restenosis after angioplasty, vein graft intimal thickening and atherogenesis. cAMP has numerous vascular protective effects, including inhibition of VSMC proliferation and migration. However, the underlying mechanisms are unknown. Here we investigated the effects of cAMPinduced cytoskeletal remodelling on the SRF co-factors, MKL1 and MKL2 and MKL-dependent transcription of pro-proliferative and migratory genes. Results: Elevation of cAMP using forskolin or dibutyryl-cAMP induced a rapid and significant reduction in cell spreading and loss of actin stress fibres in VSMC but not endothelial cells (EC). Elevated cAMP significantly inhibited mitogen-induced nuclear translocation of MKL1 and 2 in VSMC. cAMP-mediated inhibition of MKL-nuclear localisation was reversed by expression of constitutively active RhoA or sequestration of monomeric actin using cytochalasin-D. Elevated cAMP also significantly inhibited SREdependent reporter gene activity and mRNA expression of putative MKLdependent genes (CCN1 and CTGF) in VSMC but not ECs. Furthermore, cAMP-mediated inhibition of CCN1 and CTGF was reversed by forced expression of constitutively-active MKL1 (MKL1-DN100). Treatment with the MKL1/2 pharmacological inhibitor, CCG-1423 significantly inhibited VSMC proliferation and migration. Conclusions: Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling blocks MKL1 and 2 nuclear translocation and causes downregulation of pro-proliferative and pro-migratory genes in VSMC but not in EC. This mechanism contributes towards the cell type specific anti-mitogenic and anti-migratory effects and cAMP.
EFFECT OF DECELLULARIZATION PROTOCOL OF HUMAN SAPHENOUS VEINS ON CYTOTOXICITY AND MATRIX COMPONENT N. Sulaiman 1, 2, *, S.J. George 1, M.-S. Suleiman 1, R. Ascione 1. 1 School of Clinical Sciences, University of Bristol, RFLS, BRI, Bristol BS2 8HW, UK; 2 Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Cheras, Kuala Lumpur, Malaysia * Presenting author. Rationale for the study: It is proposed that development of arterial-like vascular conduits using decellularised venous extracellular matrix scaffolds seeded with cells are desirable to overcome the problem of early vein graft thrombosis, late vein graft thickening, and artificial grafts infection of autologous, xeno- and artificial graft conduit used for coronary artery bypass surgery. The aim of this study was to compare the effect of different decellularisation protocols on vein matrix components and cytotoxicity on seeded cells. Methods: Segment of human saphenous vein were decellularized by using Sodium Dodecyl Sulfate (SDS) of various percentage (0.01%, 0.025%, 0.05%, 0.075% and 0.1%). Residual cellular and extracellular matrix composition was studied with histological staining; H&E and EVG. To assess cytotoxicity and biocompatibility of the decellularised scaffold, Human Adipose Derived Stem Cells (hADSC), Human Umbilical Vein Endothelial Cells (HUVEC) and Human Smooth Muscle Cells (hSMC) were seeded and cultured on decellularised vein segments. Results: All concentration of SDS resulted in complete decellularisation of the vein without detrimental loss of collagen and elastin content and morphology. 50 000 cells was seeded on disc of decellullarised veins (4mm in diameter; 0.01% SDS; n ¼ 6) where proliferation occurred after 24 hours
e12
Abstracts / Atherosclerosis 244 (2016) e1ee12
and significantly 2-fold higher numbers of hSMC proliferation was detected compared to ADSC. Conclusions: The decellularization protocol was efficient and did not affect the extracellular matrix of veins and was observed to be non-cytotoxic to ADSCs, HUVECs and hSMCs.
PROLINE-RICH HOMEODOMAIN AND PROTEIN KINASE CK2 AS MEDIATORS OF VASCULAR SMOOTH MUSCLE CELL PROLIFERATION AND PATHOPHYSIOLOGICAL NEOINTIMA FORMATION K.S. Wadey 1, 2, *, P.-S. Jayaraman 3, K. Gaston 2, S.J. George 1. 1 School of Clinical Sciences, University of Bristol, Research Floor Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK; 2 Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK; 3 Division of Immunity and Infection, College of Medicine, University Birmingham, Edgbaston, Birmingham B15 2TT, UK * Presenting author. Saphenous vein bypass grafts display poor patency rates yet remain a much favoured surgical intervention for patients with coronary artery disease. Intimal VSMC accumulation frequently accounts for this predisposition to failure; hence, anti-proliferative therapies are an attractive approach to suppressing intimal thickening and thereby lowering incidences of restenosis. The transcription factor, proline-rich homeodomain (PRH), and its upstream regulator protein kinase CK2 have been documented to participate in mitogenic signal transduction, and aberrant expression or activity occurs in multiple pathologies. Thus far, their role in VSMC replication and intimal thickening has not been fully elucidated. We demonstrated that introduction of the PRH transgene, and silencing or pharmacological inhibition of CK2 with 10mM K66 effectively retarded proliferation in isolated rat aortic VSMCs by approximately 40%, 70%, and 50%, respectively. Moreover, the S163C:S177C PRH mutant with putative, enhanced stability exhibited a prolonged anti-mitotic action with respect to wild-type PRH when ectopically overexpressed in cultured VSMCs. Interestingly, PRH was up-regulated with neointima formation in vivo in rat carotids injured via balloon catheterisation, implying that expression may be induced to suppress excessive, uncontrolled proliferation. Importantly, gene transfer of PRH or inhibition of CK2 activity with K66 did not notably influence cell cycle progression or cell survival in cultured
HUVECs or HSaVECs, indicating that such an intervention may not be detrimental to re-endothelialisation. Finally, adenovirus-mediated overexpression of S163C:S177C PRH and treatment with K66 significantly impaired neointima formation in human saphenous vein organ cultures (10.7 ± 1.2 vs. 4.2 ± 0.9mm, n ¼ 5, p < 0.01; 39.5 ± 5.3 vs. 15.7 ± 3.5mm, n ¼ 6, p < 0.01, respectively, paired Student’s t-test) demonstrating their potential in preventing failure of vein grafts.
ACUTE SHEAR STRESS ACTIVATES NF-ҠB CANONICAL PATHWAY AND PROMOTE INFLAMMATION IN ENDOTHELIAL CELLS A.O. Ward, G.D. Angelini, S.J. George, M. Zakkar. School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol, Bristol, BS2 8HW, UK The long saphenous vein is the most used conduit in cardiac surgery, however, its use is complicated by late vein graft failure due to the development of inflammation, intimal hyperplasia and accelerated atherosclerosis. It has been demonstrated that the acute changes in shear stress when they are implanted into arterial circulation can result in significant pro-inflammatory activation mediated by p38 MAPK. There is limited data on the impact of such changes on other signalling pathways such as NF-ҠB. We hypothesise that acute shear stress can lead to the activation of canonical NF-ҠB signalling pathways in venous endothelial cells. Using Human umbilical venous endothelial cells (HUVECs) cultured either in static condition or exposed to acute shear stress at 12 dyn/cm2 for different time-points we noted that acute shear stress can lead to the upregulation of different pro-inflammatory genes as previously demonstrated. Furthermore; the exposure of HUVECs to acute shear stress for 30 and 90 min resulted in significant nuclear translocation of NF-ҠB in cells cultured under shear compared to static (p < 0.05). Such translocation was noted to be associated with significant IkBa degradation (p < 0.05). Interestingly, an increase in NF-ҠB regulatory gene, A20, was observed at 90 minutes (p < 0.05) which may suggest a rapid self-regulatory mechanism after initial exposure to shear stress. Our data suggest that the canonical NF-ҠB pathway is activated following the exposure of venous endothelial cells to high acute shear stress. Inhibition of this classical pathway might be an interesting prospect as a pretreatment for vein grafts prior to implantation.
The proven involvement of adaptive immunity, coupled with a phenotypic switch from autoimmune suppressive tolerogenic Treg to pro-inflammatory CD4+ Th1 and CD8+ T cells, during progression of obesity necessitates the presence of a triggering antigen as an activator of T and B cells. HSP60 is an evolutionary conserved mitochondrial chaperonin which can be located in the cytosol or exposed on the cell membrane also. An increase in cell membrane HSP60, which may be accompanied by HSP60 release into circulation, is especially considered a signal of autoimmunity. HSP60 is linked to obesity as a mediator of adipose tissue inflammation and insulin resistance. Moreover, circulating HSP60 levels are found to be higher in obese individuals than lean controls1. We observed an adaptive immune response against HSP60 at both T cell and B cell (antibody) levels during continuous high fat feeding of C57bl6 mice. Hence, HSP60 appears to be one of the mystery auto-antigens triggering the early T and B cell responses during obesity. Furthermore, we attempted a peptide therapy in a dose escalation protocol aiming to downregulate the inflammatory related adverse effects of obesity by achieving tolerance in T cell populations and suppressing the pathogenic antibody response. Using previously defined immuno-dominant murine HSP60 peptide combinations, we observed a significant improvement in insulin resistance (p ¼ 0.002, 2-way ANOVA) and circulating LDL/VLDL level (p ¼ 0.028). We also observed some improvement in glucose tolerance at borderline significance (p ¼ 0.057), which encourages further research to improve peptide therapy. 1 € €de A, Kriebel J, Ouwens DM, et al. Heat Marker T, Sell H, Zillessen P, Glo shock protein 60 as a mediator of adipose tissue inflammation and insulin resistance. Diabetes. 2012;61(3):615e25.
THE PLANT EXTRACT MOMORDICA CHARANTIA REDUCES EXPRESSION OF VASCULAR CALCIFICATION MARKERS IN A SMOOTH MUSCLE CELL CULTURE MODEL G.P. Sidgwick 1, *, R. Weston 1, N. Ahmad 1, N. Nazhad 1, A. Schiro 2, 3, F. Serracino Inglott 1, 2, 3, F.L. Wilkinson 1, M.Y. Alexander 1, 2. 1 Healthcare Science Research Centre, Manchester Metropolitan University, Chester Street, Manchester, M1 5GD, UK; 2 Cardiovascular Research Institute, University of Manchester, Grafton St, Manchester, Manchester Academic Health Science Centre, UK; 3 Vascular Unit, Central Manchester NHS Trust, Manchester Royal Infirmary, UK * Presenting author. The onset of vascular calcification in type-2 diabetic patients is one of the major health burdens in the industrialised world due to the associated increase in comorbidities and mortality. The fruits of the subtropical vine Momordica Charantia are thought to have potent anti-diabetic effects due to the anti-hyperglycaemic, anti-oxidant and anti-inflammatory properties of its 200+ constituents, some of which may mimic the effect of insulin. The aim of this study was to investigate the inhibitory effect of Momordica Charantia extract on vascular calcification pathways in a cell culture model. Vascular smooth muscle cells (VSMC) were isolated and cultured from femoral arteries following amputation from diabetic patients. Osteogenic differentiation was induced using b-glycerophosphate (b-GP), methylglyoxal (MGO) and additional advanced glycated end-products (AGEs). The cells were subsequently treated with varying concentrations of Momordica Charantia extract to determine the anti-calcification effects in vitro using qPCR, Alkaline phosphatase (ALP) activity and Alizarin red staining. We demonstrate that Momordica Charantia reduced gene expression of a range of biomarkers linked with vascular calcification after 4 days in a dose-dependant manner, including osteocalcin, BMP-2, C-MET and NOX-1, compared with osteogenic controls. ALP activity was also reduced in treated cells compared with osteogenic controls. Momordica Charantia extract shows promise as a potential therapeutic intervention to reduce calcification. Several small-scale clinical studies have shown the benefit of dietary supplementation in reducing the effects of diabetes, however further work is required to identify the specific component responsible for the effects, elucidate the mechanisms involved and validate the response in vitro.
e11
CYTOSKELETAL REMODELLING AND INHIBITION OF MKL DEPENDENT TRANSCRIPTION UNDERLIES THE ANTI-MITOGENIC EFFECTS OF CAMP IN SMOOTH MUSCLE CELLS M.C. Smith*, A. Duggirala, T.E. Kimura, G.B. Newby, A.C. Newby, M. Bond. School of Clinical Sciences, University of Bristol, UK * Presenting author. Background: Vascular smooth muscle cell (VSMC) proliferation contributes to restenosis after angioplasty, vein graft intimal thickening and atherogenesis. cAMP has numerous vascular protective effects, including inhibition of VSMC proliferation and migration. However, the underlying mechanisms are unknown. Here we investigated the effects of cAMPinduced cytoskeletal remodelling on the SRF co-factors, MKL1 and MKL2 and MKL-dependent transcription of pro-proliferative and migratory genes. Results: Elevation of cAMP using forskolin or dibutyryl-cAMP induced a rapid and significant reduction in cell spreading and loss of actin stress fibres in VSMC but not endothelial cells (EC). Elevated cAMP significantly inhibited mitogen-induced nuclear translocation of MKL1 and 2 in VSMC. cAMP-mediated inhibition of MKL-nuclear localisation was reversed by expression of constitutively active RhoA or sequestration of monomeric actin using cytochalasin-D. Elevated cAMP also significantly inhibited SREdependent reporter gene activity and mRNA expression of putative MKLdependent genes (CCN1 and CTGF) in VSMC but not ECs. Furthermore, cAMP-mediated inhibition of CCN1 and CTGF was reversed by forced expression of constitutively-active MKL1 (MKL1-DN100). Treatment with the MKL1/2 pharmacological inhibitor, CCG-1423 significantly inhibited VSMC proliferation and migration. Conclusions: Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling blocks MKL1 and 2 nuclear translocation and causes downregulation of pro-proliferative and pro-migratory genes in VSMC but not in EC. This mechanism contributes towards the cell type specific anti-mitogenic and anti-migratory effects and cAMP.
EFFECT OF DECELLULARIZATION PROTOCOL OF HUMAN SAPHENOUS VEINS ON CYTOTOXICITY AND MATRIX COMPONENT N. Sulaiman 1, 2, *, S.J. George 1, M.-S. Suleiman 1, R. Ascione 1. 1 School of Clinical Sciences, University of Bristol, RFLS, BRI, Bristol BS2 8HW, UK; 2 Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Cheras, Kuala Lumpur, Malaysia * Presenting author. Rationale for the study: It is proposed that development of arterial-like vascular conduits using decellularised venous extracellular matrix scaffolds seeded with cells are desirable to overcome the problem of early vein graft thrombosis, late vein graft thickening, and artificial grafts infection of autologous, xeno- and artificial graft conduit used for coronary artery bypass surgery. The aim of this study was to compare the effect of different decellularisation protocols on vein matrix components and cytotoxicity on seeded cells. Methods: Segment of human saphenous vein were decellularized by using Sodium Dodecyl Sulfate (SDS) of various percentage (0.01%, 0.025%, 0.05%, 0.075% and 0.1%). Residual cellular and extracellular matrix composition was studied with histological staining; H&E and EVG. To assess cytotoxicity and biocompatibility of the decellularised scaffold, Human Adipose Derived Stem Cells (hADSC), Human Umbilical Vein Endothelial Cells (HUVEC) and Human Smooth Muscle Cells (hSMC) were seeded and cultured on decellularised vein segments. Results: All concentration of SDS resulted in complete decellularisation of the vein without detrimental loss of collagen and elastin content and morphology. 50 000 cells was seeded on disc of decellullarised veins (4mm in diameter; 0.01% SDS; n ¼ 6) where proliferation occurred after 24 hours
e12
Abstracts / Atherosclerosis 244 (2016) e1ee12
and significantly 2-fold higher numbers of hSMC proliferation was detected compared to ADSC. Conclusions: The decellularization protocol was efficient and did not affect the extracellular matrix of veins and was observed to be non-cytotoxic to ADSCs, HUVECs and hSMCs.
PROLINE-RICH HOMEODOMAIN AND PROTEIN KINASE CK2 AS MEDIATORS OF VASCULAR SMOOTH MUSCLE CELL PROLIFERATION AND PATHOPHYSIOLOGICAL NEOINTIMA FORMATION K.S. Wadey 1, 2, *, P.-S. Jayaraman 3, K. Gaston 2, S.J. George 1. 1 School of Clinical Sciences, University of Bristol, Research Floor Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK; 2 Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK; 3 Division of Immunity and Infection, College of Medicine, University Birmingham, Edgbaston, Birmingham B15 2TT, UK * Presenting author. Saphenous vein bypass grafts display poor patency rates yet remain a much favoured surgical intervention for patients with coronary artery disease. Intimal VSMC accumulation frequently accounts for this predisposition to failure; hence, anti-proliferative therapies are an attractive approach to suppressing intimal thickening and thereby lowering incidences of restenosis. The transcription factor, proline-rich homeodomain (PRH), and its upstream regulator protein kinase CK2 have been documented to participate in mitogenic signal transduction, and aberrant expression or activity occurs in multiple pathologies. Thus far, their role in VSMC replication and intimal thickening has not been fully elucidated. We demonstrated that introduction of the PRH transgene, and silencing or pharmacological inhibition of CK2 with 10mM K66 effectively retarded proliferation in isolated rat aortic VSMCs by approximately 40%, 70%, and 50%, respectively. Moreover, the S163C:S177C PRH mutant with putative, enhanced stability exhibited a prolonged anti-mitotic action with respect to wild-type PRH when ectopically overexpressed in cultured VSMCs. Interestingly, PRH was up-regulated with neointima formation in vivo in rat carotids injured via balloon catheterisation, implying that expression may be induced to suppress excessive, uncontrolled proliferation. Importantly, gene transfer of PRH or inhibition of CK2 activity with K66 did not notably influence cell cycle progression or cell survival in cultured
HUVECs or HSaVECs, indicating that such an intervention may not be detrimental to re-endothelialisation. Finally, adenovirus-mediated overexpression of S163C:S177C PRH and treatment with K66 significantly impaired neointima formation in human saphenous vein organ cultures (10.7 ± 1.2 vs. 4.2 ± 0.9mm, n ¼ 5, p < 0.01; 39.5 ± 5.3 vs. 15.7 ± 3.5mm, n ¼ 6, p < 0.01, respectively, paired Student’s t-test) demonstrating their potential in preventing failure of vein grafts.
ACUTE SHEAR STRESS ACTIVATES NF-ҠB CANONICAL PATHWAY AND PROMOTE INFLAMMATION IN ENDOTHELIAL CELLS A.O. Ward, G.D. Angelini, S.J. George, M. Zakkar. School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol, Bristol, BS2 8HW, UK The long saphenous vein is the most used conduit in cardiac surgery, however, its use is complicated by late vein graft failure due to the development of inflammation, intimal hyperplasia and accelerated atherosclerosis. It has been demonstrated that the acute changes in shear stress when they are implanted into arterial circulation can result in significant pro-inflammatory activation mediated by p38 MAPK. There is limited data on the impact of such changes on other signalling pathways such as NF-ҠB. We hypothesise that acute shear stress can lead to the activation of canonical NF-ҠB signalling pathways in venous endothelial cells. Using Human umbilical venous endothelial cells (HUVECs) cultured either in static condition or exposed to acute shear stress at 12 dyn/cm2 for different time-points we noted that acute shear stress can lead to the upregulation of different pro-inflammatory genes as previously demonstrated. Furthermore; the exposure of HUVECs to acute shear stress for 30 and 90 min resulted in significant nuclear translocation of NF-ҠB in cells cultured under shear compared to static (p < 0.05). Such translocation was noted to be associated with significant IkBa degradation (p < 0.05). Interestingly, an increase in NF-ҠB regulatory gene, A20, was observed at 90 minutes (p < 0.05) which may suggest a rapid self-regulatory mechanism after initial exposure to shear stress. Our data suggest that the canonical NF-ҠB pathway is activated following the exposure of venous endothelial cells to high acute shear stress. Inhibition of this classical pathway might be an interesting prospect as a pretreatment for vein grafts prior to implantation.