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Human saphenous vein organ culture under controlled hemodynamic conditions.

Miyakawa AA, Dallan LA, Lacchini S, Borin TF, Krieger JE - Clinics (Sao Paulo) (2008)

Bottom Line: Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively.Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis.The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

View Article: PubMed Central - PubMed

Affiliation: Heart Institute (InCor) and Department of Medicine-LIM 13, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil. ayumi.miyakawa@incor.usp.br

ABSTRACT

Introduction: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics.

Objective: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions.

Methods: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively.

Results: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis.

Conclusion: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

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Related in: MedlinePlus

MTT stain of human saphenous vein cultured in the ex vivo perfusion system. The segments were cultured under venous (C, E, G) and arterial (D, F, H) hemodynamic conditions for 1 (C, D), 2 (E, F) or 4 (G, H) days. The freshly excised tissue (A) and dead samples (B) were used as positive and negative controls for the visualization of tissue viability. 1.5X magnification
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f5-18-0106: MTT stain of human saphenous vein cultured in the ex vivo perfusion system. The segments were cultured under venous (C, E, G) and arterial (D, F, H) hemodynamic conditions for 1 (C, D), 2 (E, F) or 4 (G, H) days. The freshly excised tissue (A) and dead samples (B) were used as positive and negative controls for the visualization of tissue viability. 1.5X magnification

Mentions: Tissue viability of the segments was examined by MTT staining (Figure 5). A control experiment was performed using a freshly isolated saphenous segment and a dead, refrigerated vessel. Since MTT stains only living cells, the fresh cross-section was completely stained, while no staining was observed in the dead vein segment (Figures 5A and 5B). The segments cultured under venous conditions from 1 to 4 days contained viable cells throughout the vessel wall, but few viable cells were verified in segments cultured under arterial conditions for 4 days (Figure 5H). To examine if the decreased cell density and viability were due to apoptotic events, a TUNEL assay was performed to detect in situ DNA fragmentation (Figures 6 and 7). Almost no apoptotic cells were observed in the saphenous vein cultured under venous conditions for up to 4 days. In contrast, cultured segments maintained for 1 and 2 days under arterial hemodynamic stimuli showed portions with several TUNEL-positive cells (Figures 6B and 6D), suggesting that the cellular losses observed in the segments cultured for 4 days may have been a result of apoptosis.


Human saphenous vein organ culture under controlled hemodynamic conditions.

Miyakawa AA, Dallan LA, Lacchini S, Borin TF, Krieger JE - Clinics (Sao Paulo) (2008)

MTT stain of human saphenous vein cultured in the ex vivo perfusion system. The segments were cultured under venous (C, E, G) and arterial (D, F, H) hemodynamic conditions for 1 (C, D), 2 (E, F) or 4 (G, H) days. The freshly excised tissue (A) and dead samples (B) were used as positive and negative controls for the visualization of tissue viability. 1.5X magnification
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664728&req=5

f5-18-0106: MTT stain of human saphenous vein cultured in the ex vivo perfusion system. The segments were cultured under venous (C, E, G) and arterial (D, F, H) hemodynamic conditions for 1 (C, D), 2 (E, F) or 4 (G, H) days. The freshly excised tissue (A) and dead samples (B) were used as positive and negative controls for the visualization of tissue viability. 1.5X magnification
Mentions: Tissue viability of the segments was examined by MTT staining (Figure 5). A control experiment was performed using a freshly isolated saphenous segment and a dead, refrigerated vessel. Since MTT stains only living cells, the fresh cross-section was completely stained, while no staining was observed in the dead vein segment (Figures 5A and 5B). The segments cultured under venous conditions from 1 to 4 days contained viable cells throughout the vessel wall, but few viable cells were verified in segments cultured under arterial conditions for 4 days (Figure 5H). To examine if the decreased cell density and viability were due to apoptotic events, a TUNEL assay was performed to detect in situ DNA fragmentation (Figures 6 and 7). Almost no apoptotic cells were observed in the saphenous vein cultured under venous conditions for up to 4 days. In contrast, cultured segments maintained for 1 and 2 days under arterial hemodynamic stimuli showed portions with several TUNEL-positive cells (Figures 6B and 6D), suggesting that the cellular losses observed in the segments cultured for 4 days may have been a result of apoptosis.

Bottom Line: Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively.Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis.The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

View Article: PubMed Central - PubMed

Affiliation: Heart Institute (InCor) and Department of Medicine-LIM 13, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil. ayumi.miyakawa@incor.usp.br

ABSTRACT

Introduction: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics.

Objective: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions.

Methods: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively.

Results: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis.

Conclusion: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Show MeSH
Related in: MedlinePlus