The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis.

OBJECTIVES: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect of beta-carotene (BC) on human umbilical cord-originated endothelial progenitor cells and human umbilical vein endothelial cells. METHODS: BC uptake was measured using high-performance liquid chromatography. The effect on cell proliferation was measured based on bromodeoxyuridine incorporation. Chemotaxis was performed in a Boyden chamber. The influence on tubular-like structure formation was investigated using a three-dimensional assay in Matrigel (Becton Dickinson, USA) both in an in vitro and in vivo model. Changes in gene expression were analyzed using the microarray hybridization method. Quantitative gene expression was estimated using the real-time polymerase chain reaction method. RESULTS: It was shown that BC, in the physiological range of concentrations found in human blood, is a potent activator of chemotaxis of endothelial cells. Microarray data analysis revealed that the genes involved in cell-cell and cell-matrix adhesion, matrix reorganization and activation of chemotaxis were the most affected by BC genes in human umbilical vein endothelial cells and endothelial progenitor cells. These results were also confirmed in an in vivo angiogenesis model. CONCLUSION: BC, in the range of physiological concentrations, stimulates early steps of angiogenic activity of endothelial cells by activation of cellular migration, as well as by matrix reorganization and a decrease in cellular adhesion.

Beta-carotene suppresses UVA-induced HO-1 gene expression in cultured FEK4.

The ultraviolet region of sunlight causes a significant oxidative stress to human skin cells and modulates expression of a series of genes in dermal fibroblasts and other cell types. The human heme oxygenase 1 (HO-1) gene is strongly activated within the first hours that follow UVA irradiation of normal human dermal fibroblasts (FEK4) and this response is being used as a marker of oxidative stress in cells. It has been shown that the induction of this gene occurs via singlet oxygen ((1)O(2)) produced upon interaction of UVA radiation with an as yet undefined cellular chromophore. Carotenoids, as the most potent singlet oxygen quenchers in nature, are expected to effectively suppress the UVA-induced HO-1 gene activation in human cells. In this study, we measured the suppression of UVA-induced levels of HO-1 mRNA after the addition of a series of six all-trans-beta-carotene concentrations (0.07, 0.2, 0.8, 2.3, 8.0, and 21 microM) to the culture medium of exponentially growing FEK4 cells. The corresponding levels of beta-carotene uptake and apo-carotenal formation were measured following HPLC separation. The results of this study show a concentration-dependent suppression of UVA- (250 kJ/m(2)) induced transcriptional activation of HO-1 in exponentially growing FEK4 cells by beta-carotene. Suppression occurred at concentrations that have been observed in human plasma after dietary supplementation with beta-carotene.

Angiogenesis in Balb/c mice under beta-carotene supplementation in diet.

Angiogenesis is a process of new blood vessel formation from pre-existing ones. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of vascular wall cells, which are mainly endothelial cells and their progenitors. The study focused on the effect of beta-carotene (BC) supplementation (12,000 mg/kg) in the diet on angiogenesis in Balb/c mice. Female Balb/c mice were fed for 5 weeks with two different diets: with BC or without BC supplementation. After 4 weeks of feeding, Balb/c mice were injected subcutaneously with two matrigel plugs with or without basic fibroblast growth factor (bFGF). Six days later, the animals were killed, and the matrigel plugs were used for immunohistochemical staining with CD31 antibody and for gene expression analysis. Microarray and Real-Time PCR data showed down-regulation of genes involved in proliferation and up-regulation of genes encoding inhibitors of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC proangiogenic activity (with or without bFGF) in vivo seemed to be more significantly associated with cells' protection from apoptosis and their stimulation of chemotaxis/homing than cell proliferation.

Human intestinal and lung cell lines exposed to beta-carotene show a large variation in intracellular levels of beta-carotene and its metabolites.

Although in vitro models are often used in beta-carotene research, knowledge about the uptake and metabolism of beta-carotene in cell lines is lacking. We measured by HPLC the intracellular levels of beta-carotene and its metabolites in 9 human intestinal and lung cell lines after exposure to 1 microM beta-carotene during 2, 6, 30, 54 h, and 3 weeks. In three colorectal carcinoma cell lines only low levels of beta-carotene could be detected and an apparent linear increase in intracellular beta-carotene was observed during the whole exposure period of 3 weeks. The remaining cell lines (an SV40 transformed colon cell line, a small intestinal carcinoma cell line and several lung cell lines) had medium or high intracellular beta-carotene levels. In these cell lines intracellular beta-carotene quickly increased during the first 54 h of exposure and after 3 weeks no further increase was observed, suggesting a stable level of beta-carotene after 54 h. Estimated intracellular concentrations at steady-state levels varied between 2 and 5 microM (low) or 9 and 55 microM (medium/high). Our results seem to indicate that an active uptake mechanism of beta-carotene exists in at least a subset of cell lines. Seven different beta-carotene metabolites were detected in the various cell lines (cis-carotene, retinol, three epoxy-carotenes, and two retinyl esters). Metabolite levels were the highest in cells with medium or high beta-carotene levels. Each cell line appeared to have a distinct metabolite profile. No intestinal or lung specific pattern could be found, but two epoxy-carotene metabolites were not detectable in the colon cell lines.