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In inflammatory diseases, tissue damage is critically associated with nitric oxide ([sup.*]NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on [sup.*]NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of [sup.*]NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of I[kappa]B kinase [beta] (IKK[beta]), I[kappa]B, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun N[H.sub.2]-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1[beta], and IL-6 but not tumor necrosis factor (TNF)-[alpha]. Tetrahydrobiopterin (B[H.sub.4]), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (B[H.sub.2]). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased B[H.sub.4]-to-B[H.sub.2] ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for B[H.sub.4] biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced 'NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited [sup.*]NO production, iNOS, and COX-2 upregulation, activation of NFKB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced [sup.*]NO production in macrophages by suppressing nuclear factor-[kappa]B activation and B[H.sub.4] biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation. roscovitine; nitric oxide; tetrahydrobiopterin; nuclear factor KB; macrophage

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91(phox) and p47(phox), NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis.

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

In inflammatory diseases, tissue damage is critically associated with nitric oxide ((*)NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (*)NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (*)NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (*)NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (*)NO production, iNOS, and COX-2 upregulation, activation of NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.