EPC-CM effects on total sprout length of <i>ex vivo</i> aortic rings are AKT and ERK dependent.

<p>Quantitative analysis of vascular outgrowth from rat aortic rings embedded in growth factor reduced-Matrigel. Incubation with EPC-CM (CM) enhanced total sprout length from the aortic rings compared to control medium incubation (Ctr). This effect was significantly abolished by addition of [5 µM] LY294002 (CM+LY) as well as of [10 µM] PD98509 (CM+PD). Data are given as mean + s.e.m. and values are presented as percentage of control. *: p < 0.05.</p

EPC-CM activates AKT and ERK pathways.

<p>Representative immunoblots <b>(A)</b> and quantitative analysis <b>(B)</b> of rBCEC4 lysates incubated with vehicle (Ctr) or EPC-CM (CM) in presence of either LY294002 (CM+LY) or PD98509 (CM+PD). Exposure of rBCEC4 to EPC-CM resulted in a marked increase of both phosphorylated AKT and phosphorylated ERK (pAKT and pERK) with regards to the total AKT and ERK levels (tAKT and tERK). Concomitant incubation with EPC-CM and the corresponding inhibitors almost completely blocked the phosphorylation of AKT and ERK. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls. *: p < 0.05.</p

Angiogenic potential of EPC-CM on tubular structure formation.

<p>Representative microphotographs of tubular network formation by microvascular endothelial cells on growth factor reduced Matrigel. EPC-CM incubation (CM) strikingly promoted the tube-like structure formation as compared to control medium (Ctr). Supplementation of EPC-CM with the inhibitor of AKT phosphorylation (CM+LY) ([5 µM]) substantially attenuated EPC-CM induced tube formation. Similarly, addition of the inhibitor of ERK phosphorylation (CM+PD) ([10 µM]) resulted in a decrease of tube formation, however, to a lesser degree as compared to the CM+LY group. Scale bar: 100 µm.</p

Basal functions of rBCEC4 are not altered by LY294002 or PD98059.

<p>Viability (A), migration (B) and capacity to organize in tubular structures (C) by rBCEC4 was not significantly changed compared to controls (Ctr) when incubated in presence of [5 µM] LY294002 or [10 µM] PD98059. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls.</p

EPC-CM effects on tubular structure formation are AKT and ERK dependent.

<p>Quantitative analysis of tubular network formation by microvascular endothelial cells on growth factor reduced Matrigel. EPC-CM incubation (CM) significantly promoted total area covered by the tube-like structures (A), total sprout length (B) and branching (C) as compared to control medium (Ctr). Supplementation of EPC-CM with the inhibitor of AKT phosphorylation (CM+LY) ([5 µM]) substantially attenuated all three parameters while the addition of the inhibitor of ERK phosphorylation (CM+PD) ([10 µM]) resulted in a significant decrease of covered area and total tube length but had no significant effect on the reduction of the number of branches. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls. *: p < 0.05.</p

Angiogenic potential of EPC-CM on <i>ex vivo</i> aortic rings.

<p>Representative microphotographs of vascular outgrowth from rat aortic rings embedded in growth factor reduced-Matrigel. Incubation with EPC-CM (CM) enhanced the formation of vascular outgrowth from the aortic rings compared to control medium incubation (Ctr). EPC-CM-enhanced capillary outgrowth was abolished by the addition of [5 µM] LY294002 (CM+LY). Similarly, incubation with EPC-CM in presence of [10 µM] PD98509 (CM+PD) significantly impaired total sprout length induced by EPC-CM. Scale bars: 200 µm and 100 µm (inserts).</p