Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells.
OBJECTIVE
The role of caveolin-1 (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis.


METHODS
Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program.


RESULTS
RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L).


CONCLUSIONS
NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti-angiogenesis therapy.
Pan
Yi-ming
YM
Department of Hepatobiliary Surgery, Drum Tower Hospital, Medical College of Nanjing University and Hepatobiliary Institute of Nanjing University, Nanjing 210008, China. panyiming6620@sohu.com
Yao
Yong-zhong
YZ
Zhu
Zhang-hua
ZH
Sun
Xi-tai
XT
Qiu
Yu-dong
YD
Ding
Yi-tao
YT
D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D051242:Caveolin 1; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester