Figure 1.
Syndecan-1 knockout increases vSMC proliferation and induces a more spread adherent cell morphology.
(A) Cell proliferation measured using an MTS assay demonstrated faster growth in syndecan-1 knockout (S1KO) vSMCs versus wild type (WT) vSMCs. (B) DNA synthesis in S1KO and WT cell lines, as indicated by the BrdU index. (C) Altered morphology of S1KO vSMCs when subjected to changes in the biochemical environment, specifically control (1% FBS), medium containing 30 µg/mL heparin, and medium containing 30 µg/mL heparin and 5 ng/mL TGF-β1. (D) Cell area after spreading was smaller for WT vSMCs in comparison to S1KO vSMCs. (E) Shape factor determinations indicated that S1KO vSMCs were more circular than WT vSMCs. (F) Elliptical form factor (EFF) determinations indicated that S1KO vSMCs were shorter and wider than their WT counterparts. Scale bar is 100 µm. *Statistically significant difference with WT group under similar culture conditions (p<0.05).
Figure 2.
Syndecan-1 knockout leads to loss of gene expression of mature vascular smooth muscle cell (vSMC) markers.
Real time PCR analyses of vSMC differentiation-related markers and tissue factor in vSMCs under the following treatments: (A) control (1% FBS), (B) medium containing 30 µg/mL heparin, and (C) medium containing 30 µg/mL heparin and 5 ng/mL TGF-β1. These analyses indicated that vSMCs were more differentiated and expressed higher levels of vSMC-specific differentiation markers in WT vSMCs relative to S1KO vSMCs. In addition, SMemb, the embryonic form of myosin heavy chain, was higher in S1KO vSMCs after treatment with heparin and TGF-β1. *Statistically significant difference with WT group under similar culture conditions (p<0.05).
Figure 3.
Measurement of protein levels of α-SMA and calponin in cultured vSMCs confirmed the upregulation of α-SMA and calponin in WT vSMCs.
(A, C) Western blotting for α-SMA and calponin in WT and S1KO cells after 48 hours of the shown treatments. (B, D) Immunostaining for α-SMA and calponin demonstrated higher expression in WT vSMCs versus S1KO vSMCs. Scale bars are 100 microns in length. *Statistically significant difference with WT group under similar culture conditions (p<0.05).
Figure 4.
Immunocytochemical analyses of the actin cytoskeleton and focal adhesions in WT and S1KO vSMCs.
(A) Paxillin in focal adhesions was more abundant and localized in large plaques near the periphery in S1KO vSMCs relative to WT. (B) Increased p-Src levels in WT relative to S1KO were observed in all media with representative images of p-Src expressed by WT and S1KO vSMCs. (C) Actin stress fibers were found to be centralized toward the nucleus in S1KO vSMCs relative to WT. (D) Increased p-paxillin levels in S1KO vSMCs was indicated by Western blotting. For all western blots, cell lysates were obtained from confluent vSMCs that were treated with control medium or with heparin or with heparin and TGF-β1 for 48 hours prior to lysis. In all Western blots, the expression levels of the target protein for S1KO vSMCs were normalized to those for WT vSMCs treated with control medium. *Statistically significant difference with WT group under similar culture conditions (p<0.05).
Figure 5.
Western blotting analysis of intracellular signaling pathways in WT and S1KO vascular smooth muscle cells (vSMCs).
For all western blots, cell lysates were obtained from vSMCs that were treated with 1% FBS, heparin or with heparin and TGF-β1 for 48 hours prior to cell lysis. (A) Western blotting for phospho-S6RP and total S6RP. (B) Immunoblotting analyses for phosphorylated PKC-α and total PKC-α. (C) Western blotting indicated reduced phospho-AKT in S1KO vSMCs versus WT vSMCs in heparin and heparin/TGF-β1 treated cells. In all quantification analyses, expression levels of the target protein for S1KO vSMCs were normalized to those for WT vSMCs treated with control culture medium. *Statistically significant difference with WT group under similar culture conditions (p<0.05).
Figure 6.
Reduced expression of calponin by aged syndecan-1 knockout (S1KO) mouse aortae relative to old wild type (WT) mice aorta.
(A) Immunohistochemical staining for calponin in the descending aorta harvested from the mice. (B) Morphometric quantification of calponin staining in the arteries (n = 5). All images were taken at the same length of exposure and calponin staining relative intensities were quantified. The intensities for S1KO mouse descending aorta were normalized relative to WT. Scale bars are 100 microns in length. *Statistically significant difference with WT (p<0.05).
Figure 7.
Treatment of vascular smooth muscle cells (vSMCs) with TNF-α alters expression of syndecan-1 (sdc-1) and the absence of syndecan-1 in syndecan-1 knockout (S1KO) increases the expression of inflammatory cytokines by vSMCs.
The cells were treated with 20/mL TNF-α for 48 hours. (A) Treatment of WT mouse cells with TNF-α reduces gene expression of sdc-1. (B) Baseline expression of IL-6 was lower in S1KO vSMCs but higher after stimulation with TNF-α. (C) Higher MCP-1 gene expression in S1KO vSMCs relative to WT vSMCs after stimulation with TNF-α. *Statistically significant difference with WT cell group under similar culture conditions. †Statistically significant difference with non-TNF-α treated WT cell group. ‡Statistically significant difference with non-TNF-α treated S1KO cell group.
Figure 8.
Inhibition of the interactions of syndecan-1 with integrins αvβ3 and αvβ5 was inhibited with the synstatin and gene or protein expression was measured after 48 hours.
(A–C) Decreased expression of vSMC-specific differentiation markers; (D–F) Increased expression of inflammatory cytokine, MCP-1, and, adhesion receptors, ICAM-1 and VCAM-1.
Figure 9.
Western blotting for increased expression of ICAM-1 and osteopontin.
*Statistically significant difference with WT group under similar culture conditions. †Statistically significant difference with non-heparin treated WT group. ‡Statistically significant differences with non-heparin treated WT/synstatin group.