SAA Promotes p38 and ERK1/2 Phosphorylation in RASMCs. Previous studies showed the pivotal role of MAPK and Akt signaling pathways in the VSMC phenotype switch [15-17].
The ERK1/2 Signaling Pathway Is Not Involved in the SAA-Induced Phenotype Switch in RASMCs. To further explore whether ERK1/2 is involved in the SAA-induced phenotype switch, U0126 (a ERK1/2 inhibitor) was introduced to block ERK1/2 phosphorylation (Figure 4(a)).
The p38 Signaling Pathway Is Involved in the SAA-Induced Phenotype Switch in RASMCs. To further explore whether p38 is involved in the SAA-induced phenotype switch, SB203580 (a p38 inhibitor) was applied to block p38 phosphorylation (Figure 5(a)).
To determine the effect of SAA on the VSMC phenotype switch, RASMCs were cultured and stimulated by SAA.
Caption: FIGURE 2: SAA promotes RASMCs toward a synthetic phenotype.
Caption: FIGURE 3: SAA promotes p38 and ERK1/2 phosphorylation in RASMCs. RASMCs were treated with 10 [micro]g/ml SAA at indicated time points.
HBMECs (3 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (3, 10 and 30 [micro]M), and RASMCs (2 x [10.sup.4] cells/well) containing different concentrations of astaxanthin (1, 3 and 10 [micro]M) were seeded in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix (BD Bioscience, San Jose, CA, USA).
HBMECs were cultured for 24 h, and RASMCs were cultured for 48 h, then washed twice with ice cold PBS on ice and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM [Na.sub.3]V[O.sub.4], 1% NP-40 and 0.02% Na[N.sub.3]) supplemented with 1 mM PMSF and 1 x protease inhibitor cocktail (Sigma, Saint Louis, MO, USA).
Astaxanthin augments proliferation, migration and tube formation HBMECs were incubated with different concentrations of astaxanthin (3-30 [micro]M) for 24 h and RASMCs were incubated with different concentrations of astaxanthin (1-10 [micro]M) for 48 h, respectively.
To directly link astaxanthin-induced signaling with angiogenesis in RASMCs, the protein expression related with Wnt/[beta]-catenin signaling pathway were assessed in RASMCs.
RASMCs (2 x [10.sup.4]) were incubated with astaxanthin 3 [micro]M for 48 h in 24-well plates in serum-free media previously coated with growth factor-reduced matrigel matrix then IWR-1-endo with 5 [micro]M continue to incubate for 8 h.
Similarly, the use of IWR-1-endo increased the expression of p-GSK3[beta], reduced the expression of wnt7a, [beta]-catenin, cyclin D1 in RASMCs, as shown in Fig.