Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells.

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

Smoking-induced monocyte dysfunction is reversed by vitamin C supplementation in vivo.

OBJECTIVE: The role of antioxidants in preventing vascular disease remains controversial. Vascular endothelial growth factor (VEGF-A) is important for endothelial and monocyte function. This study investigated the negative effects of smoking on monocyte migratory responsiveness to VEGF-A and the usefulness of vitamin C to prevent smoking-induced monocyte dysfunction. METHODS AND RESULTS: The chemotactic response of isolated monocytes from a cohort of 17 non-smokers and 10 smokers toward VEGF-A was assessed. VEGF-A significantly stimulated the migration of monocytes in non-smokers; the monocytes from smokers failed to respond to VEGF-A. Repeated analysis after 2 weeks of vitamin C intake (2 g/d) showed a fully restored VEGF-A-induced monocyte migration in smokers. VEGF-A serum levels were not altered by vitamin C. VEGF-A-inducible kinase activity was intact in monocytes from smokers as assessed by in vitro kinase assay. Monocyte dysfunction can be mimicked in vitro by challenging monocytes with a range of reactive oxygen species (ROS). CONCLUSIONS: Stimulation of monocyte migration by VEGF-A was severely attenuated in smokers, and the deficit observed was surmounted by vitamin C supplementation. The negative effects of smoking on monocyte function may translate into adverse impacts on VEGF-A-dependent repair processes such as arteriogenesis. These results propose a causative role of oxidative stress in smoking-induced monocyte dysfunction.

Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood.

OBJECTIVE: Endothelial progenitor cells (EPC) can contribute to vascular repair and targeted tumour therapy. Little is known about generating EPC from human umbilical cord blood. We therefore compared methods for purification of EPC from human umbilical cord blood. METHODS: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and used either unselected or after CD34 preselection. Unselected mononuclear cells were cultured for 9 days. Culture-dish-adherent (CDAC) and non-adherent (CDNAC) CD34+ cells were cultured separately for 4 weeks. Surface markers were assessed by immunofluorescence staining and FACS analysis. RESULTS: In unselected mononuclear cells, VEGF-R2 and VE-cadherin expression increased up to day 6. They stained positive with UEA-1 and took up acetylated LDL. Expression of CD45 and CD14 decreased over time, but remained strong. CD133 and CD34 were not expressed. CD34+-CDNAC acquired an endothelial phenotype over time with an increase of VEGFR-2 and von Willebrand factor (vWF). CD45 and CD14 decreased, while CD34 and the progenitor-cell marker CD133 remained strongly expressed. CD34(+)-CDAC showed a strong increase in VEGFR-2, CD133, CD34 and vWF, while CD14 decreased, and CD45 did not change. CONCLUSION: Putative EPC can be obtained from human umbilical cord blood. When selected for CD34, cells can be differentiated in culture to express markers of mature endothelial cells, while keeping progenitor markers. In contrast, short-term culture of unselected mononuclear cells leads to an endothelioid-monocytoid phenotype devoid of progenitor markers. Thus, the outgrowth from CD34-selected cells appears to be superior to short-term culture of unselected mononuclear cells with regard to endothelial cell-lineage specific differentiation of cells with a progenitor marker profile.

Enhanced functional response of CD133+ circulating progenitor cells in patients early after acute myocardial infarction.

AIMS: Circulating progenitor cells (PC) may contribute to myocardial recovery following infarction. Growth factors including VEGF are produced during ischaemia and stimulate PC release and activation. In this study, we focused on the functional chemotactic response of PC to VEGF in subjects early after myocardial ischaemia. METHODS AND RESULTS: Number and phenotype of PC were characterized using flow-cytometry. CD133(+)PC were isolated from peripheral blood using positive MACS isolation. The chemotactic response towards members of the VEGF family (VEGF-A, PlGF-1, and VEGF-E) was analysed in three groups: (i) early period following acute myocardial infarction (days 2-4) treated with primary PCI (AMI) (n = 35), (ii) stable coronary artery disease (CAD) (n = 35), and (iii) controls (CTR) (n = 20). CD133(+)PC number was 2-fold higher in AMI when compared with CAD and CTR (P = 0.0001), whereas CAD was not different from CTR. The chemotactic response of CD133(+)PC to VEGF-A, PlGF-1, and VEGF-E was significantly enhanced (2-fold) in AMI when compared with CAD (P = 0.0001). While the increase of the VEGFR-1-mediated/PlGF-triggered response was rapid (2 days following infarction), the VEGFR-2-mediated/VEGF-E-triggered response was maximally increased on day 4 post-AMI, thus correlating with the kinetics of maximal inflammatory activation reflected by increased CRP levels (P = 0.019). CONCLUSION: The enhanced chemotactic response of CD133(+)PC following myocardial infarction represents a novel principle potentially involved in cardiovascular repair early after myocardial infarction. Acute inflammatory processes are closely associated with this increased cellular function.