Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS(-/-) mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor N(G)-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca(2+) levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca(2+) levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol- treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca(2+) chelator, calmodulin antagonist, and CaMKKß siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca(2+)-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca(2+)/CaMKKß-dependent eNOS phosphorylation and Ca(2+)-dependent eNOS dimerization.

Angiogenic activity of sesamin through the activation of multiple signal pathways.

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125(FAK)-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.

Promotion of direct angiogenesis in vitro and in vivo by Puerariae flos extract via activation of MEK/ERK-, PI3K/Akt/eNOS-, and Src/FAK-dependent pathways.

Puerariae flos has been used for oriental herbal medicine; however, its angiogenic effect has not been elucidated. We found that the extract from Puerariae flos (PFE) increased in vitro angiogenic events, such as endothelial cell proliferation, migration, and tube formation, as well as in vivo neovascularization. These events were followed by the activation of multiple signal modulators, such as extracellular signal-regulated kinase (ERK), Akt, endothelial nitric oxide synthase (eNOS), nitric oxide production, p38, Src, and focal adhesion kinase (FAK), without increasing vascular endothelial growth factor (VEGF) expression. Inhibition of ERK, Akt, and eNOS suppressed PFE-induced angiogenic events, and inhibition of p38 and Src activities blocked PFE-induced endothelial cell migration. PFE did not affect the expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 and transendothelial permeability, which are involved in the adverse effects of the well-known angiogenic inducer VEGF. These results suggest that PFE directly stimulates angiogenesis through the activation of MEK/ERK-, phosphatidylinositol 3-kinase/Akt/eNOS-, and Src/FAK-dependent pathways, without altering VEGF expression, vascular inflammation, and permeability in vitro and in vivo and may be used as a therapeutic agent for ischemic disease and tissue regeneration.

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells.

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.