Ring Cultures Research Articles

Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1.

Regular consumption of low-dose aspirin reduces the occurrence of colorectal, esophageal, stomach, and gastrointestinal cancers. The underlying mechanism is unknown but may be linked to inhibition of angiogenesis. Because the effective doses of aspirin are consistent with the inhibition of cyclooxygenase-1 in platelets, we used liquid chromatography with tandem mass spectrometry analyses and immunoassays of human platelet releasates coupled with angiogenesis assays to search for the mediators of these effects. Blood or platelet-rich plasma from healthy volunteers stimulated with platelet activators produced a broad range of eicosanoids. Notably, preincubation of platelets with aspirin, but not with a P2Y12 receptor antagonist, caused a marked reduction in the production of 11-hydroxyeicosatetraenoic acid (HETE) and 15(S)-HETE, in addition to prostanoids such as thromboxane A2. Releasates from activated platelets caused cell migration and tube formation in cultured human endothelial cells and stimulated the sprouting of rat aortic rings in culture. These proangiogenic effects were absent when platelets were treated with aspirin but returned by coincubation with exogenous 15(S)-HETE. These results reveal 15(S)-HETE as a major platelet cyclooxygenase-1 product with strong proangiogenic effects. Thus, 15(S)-HETE represents a potential target for the development of novel antiangiogenic therapeutics, and blockade of its production may provide a mechanism for the anticancer effects of aspirin.—Rauzi, F., Kirkby, N. S., Edin, M. L., Whiteford, J. Zeldin, D. C., Mitchell, J. A., Warner, T. D. Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1.

Effect of sarcosine on endothelial function relevant to angiogenesis.

Endothelial cells (ECs) respond to changes in metabolic status and switch over to angiogenic phenotype. There are several metabolites known to mediate this transition; however, the effect of sarcosine that accumulates in invasive prostate cancer is not known. The objective of the study was to examine whether sarcosine influences EC function and affects angiogenesis. The effect of sarcosine was studied using different model systems including chick chorioallantoic membrane (CAM), rat aortic rings in culture, and human umbilical vein ECs (HUVECs) in culture. The statistical significance of difference was analyzed by one-way analysis of variance (ANOVA) and Student's t-test using GraphPad 5 software. Increased vascularization in CAM, increased endothelial sprouting in rat aortic rings in culture, and increased expression of CD31 and E-selectin suggested a possible angiogenic effect of sarcosine. Sarcosine modulated expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). In ECs in culture LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt pathway and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) reversed the effect of sarcosine. Further, sarcosine induced upregulation and activation of Akt in HUVECs. These results suggest that sarcosine modulates EC function relevant to angiogenesis through modulation of PI3K/Akt/mTOR pathway.

Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-dependent mechanism

The effect of short-term exposure to homocysteine (Hcy) on the contractile characteristics of rat aortic tissue was assessed both in vitro and in vivo. The contractile response of Hcy-treated aortic rings in culture for 1 or 4 days was unchanged from control responses. By comparison, aortic rings from animals injected with Hcy showed marked attenuation of response compared with controls injected with saline, cysteine or methionine. The contractile response to K+ was decreased within 24 hours of Hcy injection, whereas the response to both K+ (-27%) and noradrenaline (-56%) was significantly decreased by 4 days. In contrast, the contractile response to phorbol-12,13-dibutyrate was not different between Hcy and control groups. Intimal rubbing completely restored the responsiveness of Hcy-treated tissue to K+ and noradrenaline. By comparison, L-NAME only partially restored contractile responsiveness, while the cyclooxygenase inhibitor indomethacin had no effect on contractile attenuation induced by Hcy. Western blot analysis showed a 2-fold increase of endothelial nitric oxide synthase (eNOS) and a 3-fold increase in inducible nitric oxide synthase (iNOS) protein expression in the aortic endothelial cells from Hcy-injected rats. The results indicate an early detectable effect of Hcy on the in vivo contractile properties of vascular smooth muscle. The effect is endothelium-mediated and may vary depending on the agonist studied. The mechanism is uncertain but appears to involve increased nitric oxide (NO) production. Finally, the data suggest that attenuation of contraction may not be due to a direct effect of Hcy but that the compound is modified or acts indirectly in vivo.

Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-dependent mechanism

The effect of short-term exposure to homocysteine (Hcy) on the contractile characteristics of rat aortic tissue was assessed both in vitro and in vivo. The contractile response of Hcy-treated aortic rings in culture for 1 or 4 days was unchanged from control responses. By comparison, aortic rings from animals injected with Hcy showed marked attenuation of response compared with controls injected with saline, cysteine or methionine. The contractile response to K+ was decreased within 24 hours of Hcy injection, whereas the response to both K+ (-27%) and noradrenaline (-56%) was significantly decreased by 4 days. In contrast, the contractile response to phorbol-12,13-dibutyrate was not different between Hcy and control groups. Intimal rubbing completely restored the responsiveness of Hcy-treated tissue to K+ and noradrenaline. By comparison, L-NAME only partially restored contractile responsiveness, while the cyclooxygenase inhibitor indomethacin had no effect on contractile attenuation induced by Hcy. Western blot analysis showed a 2-fold increase of endothelial nitric oxide synthase (eNOS) and a 3-fold increase in inducible nitric oxide synthase (iNOS) protein expression in the aortic endothelial cells from Hcy-injected rats. The results indicate an early detectable effect of Hcy on the in vivo contractile properties of vascular smooth muscle. The effect is endothelium-mediated and may vary depending on the agonist studied. The mechanism is uncertain but appears to involve increased nitric oxide (NO) production. Finally, the data suggest that attenuation of contraction may not be due to a direct effect of Hcy but that the compound is modified or acts indirectly in vivo.

Effects of elevated P o 2 upon tracheal explants

Time-dependent effects of elevated P o 2 upon hamster tracheal rings in culture were examined. Ciliary activity of rings in 0.20 and 0.40 O 2 remained stable for 144 h. Ciliary activity decreased 25% following 120 h exposure to 0.60 O 2 and 50% within 72 h exposure to 0.95 O 2. Ciliostasis occuredd by 144 h at 0.95 O 2. [ 3H]Leucine uptake progressively decreased during 24 to 144 h exposure to 0.40, 0.60, and 0.95 O 2 when compared to 0.20 O 2. Tracheal explants incubated for 6 days under 0.20 and 0.40 O 2 had intact epithelial surface with abundant cilia, and no mucus present (scanning electron microscopy). Explants incubated under 0.60 O 2 contained mucus globules by 3 days. Incubation in 0.95 O 2 resulted in mucus globules within 3 days and epithelial sloughing, loss of cilia, and increased mucus globules within 6 days. Time and O 2 dependent increases in nuclear atypia, leukocytic infiltration into submucosal area, and focal flattening of pseudostratified ciliated epithelium were observed (light microscopy). These changes progressed following 6 days' exposure to 0.95 O 2 to complete loss of normal epithelium, with broken-up, epithelial cells along the tracheal lumen.