Characterization of Protein Expression and Signaling Pathway Activation That May Contribute to Differential Biological Functions in Porcine Arterial and Venous Smooth Muscle Cells

Figures

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  Figure 1. Differences in cell behavior between pig arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs). (A) The purity of VSMCs was demonstrated with anti-smooth muscle actin immunofluorescent staining, following the protocol described in the Materials and Methods. Scale bars = 200 microns. (B,C) Cell proliferation under the growth medium (GM) or in response to PDGF-BB stimulation was measured following the protocol described in the Materials and Methods. Different letters (a, b) indicated significant difference between two groups. (D) Cell migration assays were performed via scratch assays in serum-free (SF) or 10% FBS containing GM. (E) Migrated cells in scratched areas were counted. Scale bars = 200 microns. (F) Cells were induced to differentiate with a differentiation medium (DM), and 48 h before collecting cell lysates, PDGF-BB was applied in the indicated wells. (G,H) The densitometry of each band was obtained using Image J (NIH version 1.53K), and ratio values were obtained by interested protein bands/loading control bands. (n = 6 for calponin; n = 5 for myocardin.) ****: p < 0.000; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: no significant differences…

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  Figure 2. Differences in cell cycle proteins and extracellular matrix proteins between pig arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs). (A,C) Confluent cells were harvested and cell lysates were analyzed for the expression of the indicated proteins via immunoblots. (B,D) Individual band densitometry data were obtained using Image J (NIH version 1.53K). The ratio values were obtained by dividing the interested protein values by the loading protein values. (n = 4 for all parameters except vitronectin, which was n = 6.) (E) Cells were seeded in glass-bottom chambers and immunofluorescence staining was performed with an anti-collagen 1A1 (COL 1A1) antibody. Scale bars = 200 microns. (F) Fluorescence signals were quantified using Image J (NIH version 1.53K). ***: p < 0.001; **: p < 0.01; *: p < 0.05 were assessed using Student’s t-test.

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  Figure 3. Differential expression of focal adhesive proteins between porcine arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs). (A,C) Confluent cells were harvested and cell lysates were analyzed for the expression of indicated proteins via immunoblots. (B,D) Individual band densitometry data were obtained using Image J (NIH version 1.53K). The ratio values were obtained by dividing the interested proteins values by the loading protein values (n = 6 for integrin β3; n = 5 for ILK; n = 4 for others). (E) Cells were seeded in glass-bottom chambers and immunofluorescent staining was performed with an anti-vascular cell adhesion molecule-1 (VCAM-1) antibody. Scale bars = 200 microns. (F) Fluorescent signals were quantified using Image J (NIH version 1.53K). ****: p < 0.0001; ***: p < 0.001; *: p < 0.05; ns: non-significance were all assessed using Student’s t-test.

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  Figure 4. Differential expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between pig arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs). (A,H) Confluent cells were harvested and cell lysates were analyzed for indicated protein expression via immunoblots. (B,I) Individual band densitometry data were obtained using Image J (NIH version 1.53K). The ratio values were obtained by dividing the interested protein values by the loading protein values (n = 9 for MMP-2; n = 6 for MMP-3, MMP-9 and TIMP-3; n = 8 for TIMP-2). (C,D,F) Cells were seeded in glass-bottom chambers and immunofluorescent staining was performed with anti-MMP-9 (C), anti-MMP-3 (D) or anti-TIMP-1 (F) antibodies. Scale bars = 200 microns. (E,G) Fluorescent signals were quantified using Image J (NIH version 1.53K) for MMP-9 and MMP-3 (E) and TIMP-1 (G). ***: p < 0.001; **: p < 0.01 and ns: non-significant differences were assessed using Student’s t-test.

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  Figure 5. Differences in the activation of MAP kinases and AKT in response to PDGF-BB stimulation between pig arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs). (A,E) Confluent cells were serum-starved overnight and stimulated with PDGF-BB at the indicated time points. Cell lysates were analyzed for the expression of the indicated proteins via immunoblots. (B–D,F) Individual band densitometry data were obtained using Image J (NIH version 1.53K). The ratio values were obtained by dividing the band densitometry values of pERK1/2 (B), pJNK (C), pP38 (D) and pAKT (F) by the loading protein values (n = 4 for pERK1/2 and pJNK; n = 3 for pP38; n = 5 for pAKT). ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: non-significant differences were assessed using an ANOVA test.