Khalpey Zain, Yuen Ada H, Kalsi Kameljit K, Kochan Zdzislaw, Karbowska Joanna, Slominska Ewa M, Forni Monica, Macherini Massimo, Bacci Maria L, Batten Puspa, Lavitrano Marialuisa, Yacoub Magdi H, Smolenski Ryszard T
Heart Science Centre, Imperial College at Harefield Hospital, Middlesex UB9 6JH, UK.
Biochim Biophys Acta. 2005 Jun 30;1741(1-2):191-8. doi: 10.1016/j.bbadis.2005.03.008. Epub 2005 Apr 7.
The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.
胞外5'-核苷酸酶（E5'N，CD73）在内皮细胞表面的表达被认为对于细胞保护性、抗血栓形成和免疫抑制性腺苷的胞外形成至关重要。E5'N活性降低可能在异种移植急性血管排斥反应中起作用，阻止适应性和耐受机制。我们研究了猪心脏或内皮细胞在暴露于人类血液或血浆时E5'N活性及嘌呤代谢其他酶的变化程度，并研究了体液免疫在此过程中的作用。将野生型（WT，n = 6）和转人衰变加速因子（hDAF）基因的转基因猪心脏（T，n = 5）离体用新鲜人类血液灌注4小时。将猪主动脉内皮细胞（PAEC）暴露于自体猪血浆（PP）、正常人类血浆（NHP）或热灭活人类血浆（HHP）3小时，有或没有C1抑制剂。用基于高效液相色谱的方法在心脏或内皮细胞匀浆中测量酶活性。WT和T猪心脏中E5'N的基线活性分别为6.60±0.33 nmol/分钟/毫克蛋白和8.54±2.10 nmol/分钟/毫克蛋白（P<0.01）。用新鲜人类血液对猪心脏进行4小时的离体灌注导致WT和T心脏中E5'N活性分别降至4.01±0.32和4.52±0.52 nmol/分钟/毫克蛋白（P<0.001），尽管转基因猪的超急性排斥反应有所减轻。PAEC中E5'N的初始活性为9.10±1.40 nmol/分钟/毫克蛋白。在分别暴露于HHP和NHP 3小时后，活性分别降至6.76±0.57和4.58±0.47 nmol/分钟/毫克蛋白（P<0.01）（P<0.05），而与PP对照孵育时活性保持在9.62±0.88 nmol/分钟/毫克蛋白不变。C1抑制剂部分保留了E5'N活性，类似于HHP的作用。腺苷脱氨酶、腺苷激酶和AMP脱氨酶（嘌呤代谢的其他酶）活性呈下降趋势，但均无统计学意义。我们证明猪心脏在暴露于人类血液后E5'N活性特异性降低，这损害了腺苷的产生，导致细胞保护表型丧失，促成异种移植排斥反应。这种效应由人类体液免疫反应触发，补体起作用但未完全介导E5'N的消耗。
