β-Carotene has shown antioxidant and antiinflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of β-carotene on the production of nitric oxide (NO) and PGE2 as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-α, and IL-1β. β-Carotene inhibited the expression and production of these inflammatory mediators in both LPSstimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-κB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. β-Carotene blocked nuclear translocation of NF-κB p65 subunit, which correlated with its inhibitory effect on IκBα phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that β-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-κB activation, probably due to its antioxidant activity.

Fractalkine (FKN) has been implicated in modulation of angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We have investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increases in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells and stimulates in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, phosphatidylinositol 3-kinase (PI3K), and eNOS or transfection with dominant-negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant-negative ERK mutant blocked FKN-induced ERK, but not Akt and eNOS, phosphorylation. The PI3K inhibitor and a dominant-negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.

Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1α protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1α siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1α protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70^S6k and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1α protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1α. In addition, CORM-2 also increased stability of the HIF-1α protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1α degradation pathway. CORM-2 increased HIF-1α/HSP90α interaction, which is responsible for HIF-1α stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1α protein level, and VEGF expression. Furthermore, HSP90α knockdown suppressed CORM-2-induced increases in HIF-1α and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1α protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1α.

Prostaglandin E2(PGE2), a major product of cyclooxygenase, has been implicated in modulating angiogenesis, vascular function, and inflammatory processes, but the underlying mechanism is not clearly elucidated. We here investigated the molecular mechanism by which PGE 2 regulates angiogenesis. Treatment of human umbilical vein endothelial cells (HUVEC) with PGE 2 increased angiogenesis. PGE 2 increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), eNOS activity, and nitric oxide (NO) production by the activation of cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K). Dibutyryl cAMP (DB-cAMP) mimicked the role of PGE 2 in angiogenesis and the signaling pathway, suggesting that cAMP is a down-stream mediator of PGE 2. Furthermore, PGE 2 increased endothelial cell sprouting from normal murine aortic segments, but not from eNOS-deficient ones, on Matrigel. The angiogenic effects of PGE 2 were inhibited by the inhibitors of PKA, PI3K, eNOS, and soluble guanylate cyclase, but not by phospholipase C inhibitor. These results clearly show that PGE 2 increased angiogenesis by activating the NO/cGMP signaling pathway through PKA/PI3K/Akt-dependent increase in eNOS activity.