Inhibition of microvascular endothelial cell apoptosis by angiopoietin-1 and the involvement of cytochrome C.
BACKGROUND
Angiopoietin-1 (Ang-1) is an endothelial-specific growth factor that can promote angiogenesis. Studies demonstrated that Ang-1 can inhibit apoptosis of umbilical endothelial cells, but so far little is known about its effects on apoptosis of microvascular endothelial cells. With the apoptotic model of murine-cerebral-derived microvascular endothelial cells (bEnd.3) induced by serum-free culture, we attempted to clarify the molecular mechanism of bEnd.3 apoptosis, particularly its relation to cytochrome C (Cyt C).


METHODS
The cultured microvascular endothelial cell strain, bEnd.3 cell, was employed. An apoptotic model of bEnd.3 was established by serum-free culture. Flow cytometry after Annexin labeling and PI staining were used to assess the apoptotic effects of Ang-1 on bEnd.3, and the expression of Bax/Bcl-2, caspase 8, caspase 3, and Cyt C were detected with Western blotting and ELISA.


RESULTS
The apoptotic rate of bEnd.3 cells after stimulation with Ang-1 (100 ng/L) in serum-free medium was significantly higher than that in control group. Ang-1 inhibited early-stage apoptosis more than late-stage apoptosis provided by propidium iodide (PI) and AnnexinV double staining. The inhibition of Ang-1 on bEnd.3 cell apoptosis was strengthened with the increase in concentration (0 - 400 ng/ml). Ang-1 could decrease the expression of Bax, caspase3 and 8, and increase that of Bcl-2. The results of ELISA indicated that Ang-1 significantly decreased CytC content in cytoplasm and increase that in mitochondria.


CONCLUSIONS
Ang-1 could inhibit bEnd.3 apoptosis induced by serum-free medium culture. The apoptosis was associated with decreased Bax expression, increased Bcl-2 expression, which result in Cyt C transferring from mitochondria to cytoplasm, and then caspases activation are reduced and cell apoptosis is suppressed.
Shi
Lian-guo
LG
Department of Physiology & Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Zhang
Guo-ping
GP
Jin
Hui-ming
HM
D042683:Angiopoietin-1 / Q000494:pharmacology*; D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000032:analysis; D002478:Cells, Cultured; D045304:Cytochromes c / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D008833:Microcirculation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000032:analysis
D042683:Angiopoietin-1; D017304:Annexin A5; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases