Cleaved High Molecular Weight Kininogen Induction Of Endothelial Progenitor Cell Senescence And The Underlying Molecular Mechanism

Objective: High molecular weight kininogen (HK) is a major component of plasmakallikrein-kinin system (KKS), and is cleaved to its active form (HKa) upon theactivation of this system. The KKS activation is widely involved in a variety ofpathological settings. Thus, the understanding of vascular functions of HKa hasimportant physiological and pathological significance. Endothelial progenitor cells(EPCs) are precursors to mature endothelial cells (EC). These cells have the abilities ofpromoting reendothelialization and vascular repair. Thus, EPCs play an important rolein the maintenance of vascular homeostasis. Although a large body of evidence haveshown that HKa inhibits the function of endothelial cells, whether HKa affects thefunction of endothelial progenitor cells is unclear. In this study, we investigatedwhether HKa induced EPC senescence and explored its molecular mechanism.Methods:(1) EPCs were isolated from adult peripheral blood mononuclear cellsand expanded ex vivo.(2) To determine whether HKa affects the function of EPCs, wetested the large colony formation and proliferation of EPCs treated with50nM HKa.(3)To examine whether HKa accelerates EPC senescence, the acid β-galactosidaseactivity and telomerase activity assays were performed.(4) To explore the underlyingmechanisms, EPCs were treated with HKa at different concentrations for72hours,which were followed by the measurement of intracellular ROS generation, thephosphorylation of JNK-Thr183/Tyr185, FOXO4-Thr451and p38kinase, as wellas Mn-superoxide dismutase (MnSOD) and p16expression.(5) To narrow down thefunctional domain of HKa, recombinant proteins of human HK heavy chain (HC,19-380aa) and light chain (LC,390-644aa) were generated and used to determine whichdomain(s) mediates the effect of HKa.Results:(1) HKa markedly inhibited the large colony formation and proliferationof EPCs.(2) Treated EPCs displayed flattened and giant cell morphological changes and formation of intracellular vacuoles. As determined by acidic β-galactosidasestaining, HKa increased senescent EPCs by2-and＞3-fold after culture for1and2weeks respectively. HKa suppressed the telomerase activity of EPCs.(3) HKaconcentration-dependently increased the generation of intracellular reactive oxygenspecies (ROS), markedly enhanced phosphorylation of JNK-Thr183/Tyr185, FOXO4-Thr451and p38kinase, upregulated the expression of MnSOD and prosenescencemolecular p16at protein and mRNA levels. SB203580, a p38inhibitor, attenuatedHKa-enhanced p16expression, either quenching of ROS prevented HKa-induced EPCsenescence.(4) Similar to HKa, the heavy chain (HC) of HK, but not the lightchain(LC), increased the percentage of senescent EPCs (63±6.6%for HKa v.s.77.5±6.02%for HC). Moreover, HC at100nM increased FOXO4phosphorylation atThr451and the expression of MnSOD in EPCs. Besides, both of HKa and its HCstimulated the production of intracellular H2O2.Conclusion—These results demonstrate that HKa accelerates the onset of EPCsenescence, at least in part, by stimulating JNK/FOXO4/MnSOD/ROS/p38/p16pathway,its effect is mediated by the HC.