- Email: [email protected]
033 Co-Culture of Endothelial Progenitor Cells and Monocytes for Seeding the Surface of a Degradable Polyurethane Vascular Graft Material
S97
Abstracts
multifaceted, cell-specific, response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesize that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. METHODS: Five week old female apoE-/- mice were supplied with regular tap water (control) or drinking water supplemented with 5% glucosamine (w/v) or 5% mannitol (w/v). To induce hyperglycemia, a separate group of apoE-/- mice received multiple low dose injections of streptozotocin (STZ). All mice consumed a standard chow diet (18.8% protein, 6% fat, 3.8% fiber) and were sacrificed at 15 weeks of age for analysis. RESULTS: STZ-induced hyperglycemic and glucosamine supplemented mice had significantly larger and more advanced atherosclerotic lesions, and an increase in hepatic lipid accumulation compared to the control mice (P⬍0.05). Indications of ER stress were increased in atherosclerotic lesions and the livers of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice the accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. Treatment with either valproate or 4-phenylbutyrate, two small molecules that have been shown to block ER stress signaling, attenuated hyperglycemic and glucosamine-induced atherogenesis and hepatic steatosis. Our data suggest that elevated concentrations of glucose/glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. CONCLUSION: Similar to hyperglycemia, glucosamine-supplementation promotes ER stress, hepatic steatosis and accelerated atherosclerosis in apoE-/- mice. These findings support a model by which conditions of hyperglycemia promote hepatic and vascular complications via a glucosamine-intermediate.
033 CO-CULTURE OF ENDOTHELIAL PROGENITOR CELLS AND MONOCYTES FOR SEEDING THE SURFACE OF A DEGRADABLE POLYURETHANE VASCULAR GRAFT MATERIAL ES Mathieu, J McBane, K Battiston, JP Santerre, RS Labow, EJ Suuronen Ottawa, Ontario
The interaction of endothelial progenitor cells (EPCs) and monocytes within the blood vessel endothelium is a process of wound repair. Therefore it was of interest to investigate their function when they were cultured on a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI) material, prior to considering its use as a vascular graft. The present work investigated the cellular response of human EPCs when cultured on D-PHI films with and without monocytes. METHODS/RESULTS: Human EPCs isolated from human whole BACKGROUND:
blood, were seeded onto fibronectin-coated tissue culture polystyrene (TCPS), D-PHI films coated with fibronectin or DPHI films with or without autologous monocytes for up to 7 days. EPCs were then analyzed for viability and attachment (WST assay and DNA), as well as nitric oxide (NO) and cytokine production. Lysates of EPC cultures were analyzed by Western blot to assess EPC differentiation into an endothelial cell (EC) phenotype (CD31 expression). As indicated by a WST assay, EPCs alone remained viable over the 7-day culture period on D-PHI films (day 1 vs. day 7, 0.06⫾0.009 vs. 0.15⫾0.026 WST-1 units/200,000 cells; p⫽NS) and in coculture with monocytes (0.06⫾0.006 vs. 0.06⫾0.018 WST-1 units/200,000 cells; p⫽NS). There was no significant difference in EPC attachment to D-PHI films or in NO production when EPCs were seeded on fibronectin-coated TCPS or with monocytes (p⫽NS). CD31 expression, linked to differentiation into ECs, was increased at the 7-day time point for each condition. EPCs cultured on fibronectin-coated TCPS or DPHI films showed that EPCs were activated to a wound-healing phenotype, characterized by low TNF-␣ and elevated IL-10 levels (10.62⫾4.01 vs. 106.22⫾40.10 pg/g DNA for TNF-␣ vs. IL-10, respectively, at day 7 for EPCs on D-PHI films); and in co-culture with monocytes, levels of the inflammatory cytokine TNF-␣ were decreased (17.02⫾2.53 vs. 1.33⫾0.94; for day 1 vs. day 7, respectively). CONCLUSION: These results demonstrated that D-PHI films performed as well as fibronectin-coated TCPS in terms of the biological parameters assayed and that it was not necessary to coat the D-PHI films with fibronectin to promote EPC attachment or phenotype. Moreover, the data showed that EPCmonocyte co-culture might aid in promoting a wound-healing monocyte/macrophage phenotype. This suggests that co-culturing EPCs with autologous monocytes on D-PHI may be suitable for tissue engineering a vascular graft.
034 ENDOTHELIAL NITRIC OXIDE SYNTHASE-OVEREXPRESSING HUMAN EARLY OUTGROWTH ENDOTHELIAL PROGENITOR CELLS DECREASE HUMAN CORONARY ARTERY SMOOTH MUSCLE CELL MIGRATION THROUGH PARACRINE FUNCTIONS S Guber, T Ebrahimian, S Wassmann, S Lehoux Montréal, Québec
Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that
Abstracts
multifaceted, cell-specific, response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesize that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. METHODS: Five week old female apoE-/- mice were supplied with regular tap water (control) or drinking water supplemented with 5% glucosamine (w/v) or 5% mannitol (w/v). To induce hyperglycemia, a separate group of apoE-/- mice received multiple low dose injections of streptozotocin (STZ). All mice consumed a standard chow diet (18.8% protein, 6% fat, 3.8% fiber) and were sacrificed at 15 weeks of age for analysis. RESULTS: STZ-induced hyperglycemic and glucosamine supplemented mice had significantly larger and more advanced atherosclerotic lesions, and an increase in hepatic lipid accumulation compared to the control mice (P⬍0.05). Indications of ER stress were increased in atherosclerotic lesions and the livers of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice the accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. Treatment with either valproate or 4-phenylbutyrate, two small molecules that have been shown to block ER stress signaling, attenuated hyperglycemic and glucosamine-induced atherogenesis and hepatic steatosis. Our data suggest that elevated concentrations of glucose/glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. CONCLUSION: Similar to hyperglycemia, glucosamine-supplementation promotes ER stress, hepatic steatosis and accelerated atherosclerosis in apoE-/- mice. These findings support a model by which conditions of hyperglycemia promote hepatic and vascular complications via a glucosamine-intermediate.
033 CO-CULTURE OF ENDOTHELIAL PROGENITOR CELLS AND MONOCYTES FOR SEEDING THE SURFACE OF A DEGRADABLE POLYURETHANE VASCULAR GRAFT MATERIAL ES Mathieu, J McBane, K Battiston, JP Santerre, RS Labow, EJ Suuronen Ottawa, Ontario
The interaction of endothelial progenitor cells (EPCs) and monocytes within the blood vessel endothelium is a process of wound repair. Therefore it was of interest to investigate their function when they were cultured on a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI) material, prior to considering its use as a vascular graft. The present work investigated the cellular response of human EPCs when cultured on D-PHI films with and without monocytes. METHODS/RESULTS: Human EPCs isolated from human whole BACKGROUND:
blood, were seeded onto fibronectin-coated tissue culture polystyrene (TCPS), D-PHI films coated with fibronectin or DPHI films with or without autologous monocytes for up to 7 days. EPCs were then analyzed for viability and attachment (WST assay and DNA), as well as nitric oxide (NO) and cytokine production. Lysates of EPC cultures were analyzed by Western blot to assess EPC differentiation into an endothelial cell (EC) phenotype (CD31 expression). As indicated by a WST assay, EPCs alone remained viable over the 7-day culture period on D-PHI films (day 1 vs. day 7, 0.06⫾0.009 vs. 0.15⫾0.026 WST-1 units/200,000 cells; p⫽NS) and in coculture with monocytes (0.06⫾0.006 vs. 0.06⫾0.018 WST-1 units/200,000 cells; p⫽NS). There was no significant difference in EPC attachment to D-PHI films or in NO production when EPCs were seeded on fibronectin-coated TCPS or with monocytes (p⫽NS). CD31 expression, linked to differentiation into ECs, was increased at the 7-day time point for each condition. EPCs cultured on fibronectin-coated TCPS or DPHI films showed that EPCs were activated to a wound-healing phenotype, characterized by low TNF-␣ and elevated IL-10 levels (10.62⫾4.01 vs. 106.22⫾40.10 pg/g DNA for TNF-␣ vs. IL-10, respectively, at day 7 for EPCs on D-PHI films); and in co-culture with monocytes, levels of the inflammatory cytokine TNF-␣ were decreased (17.02⫾2.53 vs. 1.33⫾0.94; for day 1 vs. day 7, respectively). CONCLUSION: These results demonstrated that D-PHI films performed as well as fibronectin-coated TCPS in terms of the biological parameters assayed and that it was not necessary to coat the D-PHI films with fibronectin to promote EPC attachment or phenotype. Moreover, the data showed that EPCmonocyte co-culture might aid in promoting a wound-healing monocyte/macrophage phenotype. This suggests that co-culturing EPCs with autologous monocytes on D-PHI may be suitable for tissue engineering a vascular graft.
034 ENDOTHELIAL NITRIC OXIDE SYNTHASE-OVEREXPRESSING HUMAN EARLY OUTGROWTH ENDOTHELIAL PROGENITOR CELLS DECREASE HUMAN CORONARY ARTERY SMOOTH MUSCLE CELL MIGRATION THROUGH PARACRINE FUNCTIONS S Guber, T Ebrahimian, S Wassmann, S Lehoux Montréal, Québec
Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that