Pandolfi Assunta, Di Pietro Natalia, Sirolli Vittorio, Giardinelli Annalisa, Di Silvestre Sara, Amoroso Luigi, Di Tomo Pamela, Capani Fabio, Consoli Agostino, Bonomini Mario
Department of Biomorphology, University G. d'Annunzio, Aging Research Center, Ce.S.I., G. d'Annunzio University Foundation, Chieti-Pescara, Italy.
J Cell Physiol. 2007 Dec;213(3):699-709. doi: 10.1002/jcp.21138.
In end-stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC-human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro-inflammatory effects due to decreased NO bio-availability, enhanced ESRD-RBC-HUVEC interactions might directly stimulate pro-inflammatory pathways leading to increased vascular adhesion molecule expression. ESRD-RBC-endothelial cell interactions induced a time-dependent up-regulation of VCAM-1 and ICAM-1 (measured by Western blot (WB) and real-time PCR), associated with mitogen-activated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM-1 and ICAM-1 mRNA levels when incubated on HUVEC. Interestingly, ESRD-RBC induced increased expression of adhesion molecules was prevented by Annexin-V (AnV, able to mask PS on RBC surface), anti-integrin-alpha(v)beta3, anti-thrombospondin-1 (TSP-1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD-RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin-(alpha(v)beta3) integrin complex, ESRD-RBC-HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD-RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions.
在终末期肾病（ESRD）中，内皮可能是循环成分（如修饰红细胞（RBC）和/或血浆因子）作用的关键靶点，这些循环成分可能会促进炎症以及与尿毒症相关的血管病变。我们之前已经证明，ESRD患者红细胞表面磷脂酰丝氨酸（PS）暴露会增加红细胞与人脐静脉内皮细胞（HUVEC）的相互作用，并导致一氧化氮（NO）生成减少。我们推测，除了由于NO生物利用度降低而产生的促炎作用外，增强的ESRD-RBC-HUVEC相互作用可能直接刺激促炎途径，导致血管黏附分子表达增加。ESRD-RBC与内皮细胞的相互作用诱导了VCAM-1和ICAM-1的时间依赖性上调（通过蛋白质印迹法（WB）和实时PCR测量），这与丝裂原活化蛋白激酶（MAPK）激活以及通过WB测量的Akt/内皮型一氧化氮合酶（eNOS）信号级联受损有关。在重组实验中，与尿毒症血浆孵育的正常红细胞在与HUVEC孵育时显示出PS暴露增加，并且VCAM-1和ICAM-1 mRNA水平显著升高。有趣的是，膜联蛋白-V（AnV，能够掩盖红细胞表面的PS）、抗整合素-α(v)β3、抗血小板反应蛋白-1（TSP-1）和PD98059（MAPK磷酸化的选择性抑制剂）可阻止ESRD-RBC诱导的黏附分子表达增加。此外，AnV逆转了ESRD-RBC对MAPK和Akt/eNOS信号通路的影响。我们的数据表明，ESRD-RBC-HUVEC黏附可能通过与内皮血小板反应蛋白-（α(v)β3）整合素复合物的直接相互作用，诱导血管炎症表型。因此，针对ESRD-RBC增加的与内皮细胞黏附以及/或MAPK和Akt/eNOS途径的干预措施可能有潜力预防尿毒症条件下的血管病变。
