Monocytes and vascular endothelial cells apoptosis. Role of p-HSP27.
The aim of this study was to find out whether stimulated monocytes could trigger apoptosis of vascular endothelial cells. Human umbilical vein endothelial cells (HUVEC) (EC) were co-cultured for 24 h and 48 h with monocytes isolated from peripheral blood (peripheral blood monocytes) or MonoMac6 cell line activated previously with proinflammatory cytokines. Real-time PCR was conducted to investigate p53 up-regulated modulator of apoptosis (PUMA), heat shock protein HSP70 and HSP27 genes expression. Changes in the level of PUMA, HSP70, HSP27 and phospho-heat shock protein 27 (p-HSP27) proteins were analyzed by means of immunoprecipitation. Apoptosis was determined by TUNEL and poli-(ADP ribose) polymerase ( PARP ) cleavage assay. In HUVEC cells stimulated with monocytes hardly any increase of PUMA mRNA was observed, but the PUMA protein level was significantly up regulated especially after 24 h. Heat shock proteins (HSP70 and HSP27) mRNA expression was elevated after 24 h and 48h and confirmatory up regulation of these proteins was observed in HUVEC cells stimulated with peripheral blood monocytes but not with MonoMac6 cells. Interestingly, in nuclear compartment of HUVECs exposed to the monocytic line and native monocytes, a significant increase of p-HSP27 level has appeared. TUNEL and PARP cleavage assay did not show any apoptotic HUVEC cells after stimulation with monocytes. The main observations of this study indicate that monocytes do not trigger apoptosis of vascular endothelial cells. Proapoptotic activation mediated by PUMA that was observed seemed to be counterbalanced by significant increase of antiapoptotic HSP70, HSP27 and especially phospho-HSP27 proteins level.
Gawad
A
A
Department of Physiology, Jagiellonian University, Medical College, Cracow, Poland.
Ptak-Belowska
A
A
Brzozowski
T
T
Pawlik
W W
WW
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D020287:In Situ Nick-End Labeling; D018832:Molecular Chaperones; D009000:Monocytes / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D016207:Cytokines; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011065:Poly(ADP-ribose) Polymerases