Phosphorylation of GST-c-Jun was then visualized by SDS-PAGE and autoradiography imaging

GroEL1-treated EPCs. Western blot analysis showed cleaved caspase-9, caspase-8, and cleaved caspase-3 were increased under GroEL1 treatment in late EPCs. After 1 hour of preincubation with Z-VAD-FMK, Ac-LEHD-CMK, or Ac-IETD-CHO, senescence was significantly decreased in GroEL1-stimulated EPCs. These findings indicate that EPC senescence 21363929 may be mediated by caspases. Additionally, senescent EPCs express higher levels of MAPKs. Indeed, western blotting indicated that treatment with GroEL1 altered the activation of p38 MAPK and ERK1/2, which effects were inhibited by an inhibitor of p38 MAPK and an inhibitor of ERK1/2. In contrast, GroEL1 did not induce the activation of JNK/SAPK in EPCs; treatment with PD98059, a JNK/SAPK antagonist, also did not prevent senescence of EPCs, suggesting that the JNK/SAPK signaling pathways might not participate in senescence of GroEL1treated EPCs. Discussion In the present study, we provide evidence that 1) heat shock protein 60 of C. pneumonia, GroEL1, impaired the recovery of capillary density, which may be mediated by TLR4 in mice; 2) GroEL1 impaired EPC mobilization and vessel formation as well as eNOS expression in ischemic tissue; 3) GroEL1 administration impaired the migration and vasculogenesis of late EPCs in vitro; 4) EPC senescence was enhanced by GroEL1, which was mediated by activation of caspases, p38 MAPK and ERK1/2; and 5) GroEL1 decreased integrin and Eselectin expression but induced inflammatory responses in EPCs. These findings suggest that TLR4 and impaired NOrelated mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of C. pneumonia GroEL1. Our study demonstrates that a TLR4-associated MedChemExpress KU55933 mechanism contributes to neovascularization in inflammation-associated angiogenesis in ischemic tissue. Activation of the immune system via TLRs is implicated in angiogenesis, atherosclerosis, and various vascular complications. 10884437 However, the mechanisms by which immunity and inflammatory responses are involved in angiogenesis of ischemic tissue have not been elucidated completely. Recent studies highlight the critical role of TLRs in the induction of inflammatory responses in ischemic diseases. For example, deficiency of TLR4 protects the myocardium from ischemic injury, whereas modulation of TLR2 induces cardioprotection against ischemic insult. In addition, TLRs are involved in the induction of angiogenesis, modulation of stem cell function, and expression of microRNAs, which are currently important areas in ischemia research. By contract, without exogenous pathogen-associated ligands, TLR4 can be activated by endogenous agonists, such as highmobility group box 1, which is produced by damaged tissue or infiltrating immune cells in the injured sites. He et al. used an acute oxygen-induced ischemic retinopathy model in TLR4-/- and WT mice to discover an unrecognized pathway involving HMGB1 and its interaction with TLR4 in angiogenesis. Their results suggest that TLR4 deficiency retards the TLR4-mediated response and downregulates the expression of VEGF, resulting in decreased neovascularization. These results reveal a novel aspect of the multi-faceted TLR biology and may suggest new prospects for using TLR4related mechanisms to modulate the production of EPCs for clinical use. GroEL1 Decreases Integrin and E-selectin Expression and Induces Inflammatory Responses in EPCs In vitro and in vivo evidence indicates many integrins and selectins function in the regulat