Objective: In the present study, we sought to elucidate whether astaxanthin contributes to induce angiogenesis and its mechanisms.
Materials and methods: To this end, we examined the role of astaxanthin on human brain microvascular endothelial cell line (HBMEC) and rat aortic smooth muscle cell (RASMC) proliferation, invasion and tube formation in vitro. For study of mechanism, the Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo was used. HMBECs and RASMCs proliferation were tested by cell counting. Scratch adhesion test was used to assess the ability of invasion. A matrigel tube formation assay was performed to test capillary tube formation ability. The Wnt/[beta]-catenin pathway activation in HMBECs and RASMCs were tested by Western blot.
Results: Our data suggested that astaxanthin induces angiogenesis by increasing proliferation, invasion and tube formation in vitro. Wnt and f)-catenin expression were increased by astaxanthin and counteracted by IWR-1-endo in HMBECs and RASMCs. Tube formation was increased by astaxanthin and counteracted by IWR-1-endo.
Conclusions: It may be suggested that astaxanthin induces angiogenesis in vitro via a programmed Wnt/[beta]-catenin signaling pathway.
Angiogenesis, the growth of new blood vessels, is a crucial force for shaping the nervous system and protecting it from disease. Angiogenesis and neurogenesis are prominent features of neurological disease, either as pathophysiological factors or as responses to injury. Neurovascular responses have a critical role as the damaged central nervous system transitions from initial injury into repair (Zhang and Chopp, 2009). Reestablishment of functional microvasculature enhances neurogenesis and functional recovery after stroke (Dalkara et al., 2011; Pries and Secomb, 2014). Enhanced angiogenesis should provide new opportunities for stroke recovery (Zeng et al., 2014).
The Wnt/[beta]-catenin signaling plays a prominent role in cell differentiation, adhesion, survival and apoptosis, and is involved in organ development, neurogenesis, and tissue fibrosis, among other functions. [beta]-catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes (Akiyama, 2000; Satoh and Kuroda, 2000). The lack of a single [beta]-catenin allele shows already abnormal blood vessel development and leads to embryonic lethality.
Astaxanthin is a xanthophyll carotenoid and belongs to a larger class of phytochemicals known as terpenes. Natural astaxanthin is produced and extracted from Haematococcus microalgae (Lorenz and Cysewski, 2000). It has shown that astaxanthin has antioxidant and anti-inflammatory effects (Pashkow et al., 2008). It has also shown protective potential against cerebral ischemia injury (Ergul et al., 2012). In the present study, we therefore investigated the hypothesis that astaxanthin induced angiogenesis in cerebral endothelial cells and smooth muscle cells and regulated Wnt/[beta]-catenin signaling pathway through activating [beta]-catenin.
Materials and methods
Astaxanthin (purity > 98.0%, molecular formula [C.sub.40][H.sub.52][O.sub.4]: 596.86). A stock solution of astaxanthin was made in DMSO at a concentration of 30 mM. The following pharmacologic agents were used: Wnt/[beta]-catenin signaling pathway inhibitor IWR-1-endo (Calbiochem).
Human brain microvascular endothelial cell (HEMEC) culture
A human brain microvascular endothelial cell line (HBMEC) were seeded at 60%-70% confluence and kept at 37[degrees]C in 5% C[O.sub.2]. Culture...