In in vivo studies, we observed that BC injected locally subcutaneously with matrigel augmented the angiogenic response initiated by bFGF in Balb/c mice. In order to estimate the effect of BC in diet on angiogenesis in vivo, mice were fed with the diet that contained 12% of BC. of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC VO-Ohpic trihydrate 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. Keywords:Angiogenesis, Beta-carotene, Chemotaxis, Microarray == Introduction == Angiogenesis, the process of new blood vessels formation, plays a central role in both normal (physiological) and pathological events [6]. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of the endothelial and/or their progenitor cells [14]. Beta-carotene (BC), which represents provitamin VO-Ohpic trihydrate A carotenoid obtained in diet from dark green and colourful vegetables, is a source of retinoids that act as ligands for the nuclear retinoid receptors and promote cell differentiation in humans [16]. Our previous in vitro studies confirmed the proangiogenic effect of BC on angiogenesis in human umbilical endothelial cells (HUVECs) [7,15] and endothelial progenitor cells (EPCs) [9]. Physiological concentrations of BC (3 M) did not change the tubulogenic activity of HUVECs in the in vitro angiogenesis model, but BC stimulated chemotaxis of both cell types (mean ratio of stimulated migration by BC 3 M VO-Ohpic trihydrate for HUVECs was 4, for EPCs it was 6). These observations were in agreement with the microarray data, confirmed by Real-Time PCR (RT-PCR), which revealed changes in the expression of the genes that influenced cell adhesion, matrix reorganization, homing and chemotaxis. The up-regulated genes included early growth response 1 (EGR1) (cell differentiation activator), baculoviral IAP repeat-containing 3 (BIRC3) (inhibitor of apoptosis), IL8 and chemokine (CC motif) ligand 2 (CCL2) (activators of migration), CXCL12 and CXCR4 (activators of homing) [9,15]. Thus, our in vitro studies demonstrated that BCs physiological concentrations stimulate early steps of angiogenesis by the activation of cellular migration, matrix reorganization and decrease in cell adhesion. Prochemotactic and homing activity VO-Ohpic trihydrate of BC in EPC cells suggested its potential role in the physiology of progenitor Rabbit Polyclonal to EHHADH cells that might make them useful in therapy directed towards tissue repair [17]. The BC effects observed in the cell culture needed to be confirmed in vivo. Here, we investigated the effects of BC in wild type Balb/c mice using a matrigel angiogenesis model. == Materials and methods == == Mice == The protocol was accepted by the Jagiellonian University ethics committee. To investigate the effect of BC (12,000 mg/kg in the diet) on angiogenesis, 8 week old female Balb/c mice (n= 610 per group) were fed with chow diet (Kliba 2415) [Provimi Kliba AG, Switzerland containing vitamin A 1,400 U/kg] supplemented with BC beatlets [12%1,200 ppm BC] or control, no-BC beatlets (kindly supplied by DSM Neutraceuticals) for 5 weeks. In the control diet (without BC beatlets), amount of vitamin A was the same as in the diet with BC beatlets. To estimate BC intake by animals, BC concentration in serum was controlled after 5 weeks of experimental feeding with the HPLC micromethod developed VO-Ohpic trihydrate by DSM neutraceuticals (Roche Vitamins AG) (Kaiseraugust, Switzerland) [2]. == The used model of angiogenesis == Balb/c mice received sterile abdominal injections of 2 500 L matrigel subcutaneously 4 weeks after initialization of their assigned diets. To induce angiogenesis, matrigel plug contained bFGF (50 nM) and for control no-bFGF solvent (phosphate-buffered saline with 0.5% BSA). Six days later, the animals were killed, and the matrigel plugs were removed. The paraffin embedded sections were immunohistochemically stained for CD31 (PECAM) (Becton Dickinson) antigen, a marker characteristic for endothelial cells. Number of capillaries with and without lumen along with the number of separate PECAM positive cells was counted under light microscope in five different fields in each of the three slices taken from different parts of each plug by an uninformed, trained morphologist according to the published protocols [21]. To investigate.