Impact of normothermic perfusion and protein supplementation on human endothelial cell function during organ preservation.
BACKGROUND
Hypothermia-induced changes in endothelial cell (EC) morphology and function after organ storage may influence the initial outcome and development of transplant-associated coronary artery disease.


METHODS
Human saphenous vein ECs were incubated with saline (NaCl), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solution, with and without protein additives, at 4 degrees C and 37 degrees C. After 6 hours, ECs were recultivated for 24 and 48 hours with culture medium (reperfusion). Mitochondrial activity, adenosine triphosphate concentration, cell count, and inflammatory responses were analyzed.


RESULTS
Cold preservation did not affect the mitochondrial activity of ECs and allowed a complete regeneration of the metabolic turnover after reperfusion. However, under normothermic conditions the metabolism of the cells was influenced by time and type of preservation solution. While both the mitochondrial activity and cell count did not change after treatment with NaCl and culture medium, the metabolic turnover of cells treated with HTK and UW solution significantly increased (twofold) and decreased (twofold, p < 0.05), respectively, after reperfusion. The endothelial reactivity remained unchanged after treatment with NaCl and HTK. The addition of serum proteins significantly improved mitochondrial activity of cells treated with warm NaCl and HTK (p < 0.05). The UW-treated cells burned out through a significant up-regulation of the ATP concentration resulting in a complete metabolic regression after reperfusion and induction of apoptosis.


CONCLUSIONS
Normothermic preservation in UW prevented regeneration of ECs, while treatment with HKT solution did not irreversibly affect mitochondrial activity of ECs and allowed complete regeneration of metabolism and function. Serum proteins improved the preservation effect of HTK and NaCl.
Puehler
Thomas
T
Department of Cardiothoracic Surgery, University Medical Center Regensburg, Regensburg, Germany. thomas.puehler@klinik.uni-regensburg.de
Gleich
Otto
O
Schopka
Simon
S
Rupprecht
Leopold
L
Hirt
Stephan
S
Schmid
Christof
C
Lehle
Karla
K
D000241:Adenosine; D000255:Adenosine Triphosphate / Q000378:metabolism; D000493:Allopurinol; D017209:Apoptosis / Q000502:physiology; D001798:Blood Proteins / Q000494:pharmacology*; D002448:Cell Adhesion / Q000502:physiology; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology*; D015925:Cryopreservation; D019040:E-Selectin / Q000378:metabolism; D042783:Endothelial Cells / Q000502:physiology*; D004734:Energy Metabolism / Q000502:physiology*; D005947:Glucose; D005978:Glutathione; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin; D008353:Mannitol; D008928:Mitochondria / Q000502:physiology; D009926:Organ Preservation / Q000379:methods*; D019188:Organ Preservation Solutions; D010477:Perfusion; D011189:Potassium Chloride; D011343:Procaine; D011887:Raffinose; D012965:Sodium Chloride; D052096:Warm Ischemia
D001798:Blood Proteins; C039984:Bretschneider cardioplegic solution; D019040:E-Selectin; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D008353:Mannitol; D012965:Sodium Chloride; D011343:Procaine; D000493:Allopurinol; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D005978:Glutathione; D005947:Glucose; D000241:Adenosine; D011887:Raffinose