Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells

Aim： The role of caveolin-1 （Cav-1） in angiogenesis remains poody 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 N^G-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）. Conclusion： 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.